Universal donor cells and related methods

ABSTRACT

Disclosed herein are universal donor stem cells and cells derived therefrom and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming allogeneic immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor cells disclosed herein are pancreatic endoderm cells that do not express one or more MHC-Class I cell-surface proteins and whose expression of at least one NK activating ligand is disrupted or inhibited.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 15/648,337,filed Jul. 12, 2017, which is incorporated by reference herein in itsentirety

FIELD

This relates to the fields of gene expression, genome engineering andgene/cell therapy.

BACKGROUND

Human pluripotent stem cells (hPSCs) are a useful tool to generate anyadult cell type for transplantation into patients. In principle,hPSC-based cell therapies have the potential to treat most if not alldegenerative illnesses, however the success of such therapies may belimited by a subject's immune response.

The immune system protects organisms from infection with layereddefenses of increasing specificity. In simple terms, physical barriersprevent pathogens such as bacteria and viruses from entering theorganism. If a pathogen breaches these barriers, the innate immunesystem provides an immediate, but non-specific response. If pathogenssuccessfully evade the innate response, vertebrates possess a secondlayer of protection, the adaptive immune system, which is activated bythe innate response. The adaptive immune system generates a much morespecific response. Here, the immune system adapts its response during aninfection to improve its recognition of the pathogen. This improvedresponse is then retained after the pathogen has been eliminated, in theform of an immunological memory, and allows the adaptive immune systemto mount faster and stronger attacks each time this pathogen isencountered The adaptive immune response is antigen-specific andrequires the recognition of specific “non-self” antigens during aprocess called antigen presentation. Antigen specificity allows for thegeneration of responses that are tailored to specific pathogens orpathogen-infected cells. Interferon gamma (IFN-γ) plays an essentialrole in combating infectious and non-infectious diseases. The principalsource of IFN-γ in the human immune response is T cells. NK cells,macrophages, and IFN-play an important role in both innate and acquiredimmunity.

The major histocompatibility complex (MHC) is a set of cell surfaceproteins essential for the regulation of the immune system. The mainfunction of MHC molecules is to bind to antigens derived from pathogensand display them on the cell surface for recognition by the appropriateT-cells. The MHC gene family is divided into three subgroups: class I,class II, and class III. The human MHC is also called the HLA (humanleukocyte antigen) complex (often just the HLA).

Natural killer (NK) cells are lymphocytes that function at the interfacebetween innate and adaptive immunity. NK cells contribute directly toimmune defense through their effector functions, such as cytotoxicityand cytokine secretion, and by regulating innate and adaptive immuneresponses. When a target or host cell encounters NK cells severaloutcomes are possible. The extent of the NK response is determined bythe amount and type of activating and inhibitory receptors on the NKcells and the amount and type of activating and inhibitory ligands onthe target cell. See FIG. 1. In scenario A, when target cells have nohuman leucocyte antigen (HLA) Class I and no NK activating ligands, NKcells expressing MHC-Class I inhibitory receptors and activating ligandreceptors do not attack target cells (no response, or not-licensed). Inscenario B, when target cells express HLA-Class I but have no activatingligands, the NK cells expressing inhibitory receptors and activatingreceptors cannot attack the targets. In scenario C, when target cellshave downregulated HLA-Class I or no HLA-Class I and express NKactivating ligands, NK cells expressing inhibitory receptors andactivating receptors attack target cells. In scenario D, when targetcells express both self-HLA-Class I and NK activating ligands, then thelevel of response by NK cells expressing inhibitory receptors andactivating receptors is determined by the balance of inhibitory andactivating signals to the NK cell. Haynes et al., THE IMMUNE SYSTEM INHEALTH AND DISEASE, PART 15: Immune-Mediated, Inflammatory, andRheumatologic Disorders, 372e Introduction to the Immune System.

Historically, efforts to overcome a host's immune response to allogeniccells focused on the adaptive immune response, that is, interfering withadhesion between T-cells and MHC-Class I antigens presented on foreigncells. As such, CRISPR and TALEN systems have been used to generate lossof function genetic modifications and thus make stem cells that do notexpress one or more classic MHC/HLA genes. However, these cells andcells derived therefrom are still vulnerable to the host's innate immuneresponse (NK cells). See, e.g., Parham et al. (2005) Nat Rev Immunol.5(3):201-214. In order to overcome the host's innate immune response,others have tried to reintroduce tolerogenic factors back into thetarget cell; the focus was on the “missing self.” See WO2016183041A2 thedisclosure of which is incorporated by reference in its entirety.Applicants surprisingly discovered that the key to evading the host's NKmediated immune response is not the “missing self” but the expressionand magnitude of NK cell activating ligands on target cells.

Thus, there remains a need for compositions and methods for developingtarget cells that lack some or all classic HLA expression but whichcells are not attacked by NK cells for lysis.

SUMMARY

Disclosed herein are strategies to overcome graft rejection, inparticular, allogenic immune graft rejection in cell-basedtransplantation therapies by providing universal donor cell lines. Inone embodiment, human pluripotent stem cells are provided that lack someor all classic HLA-Class I cell surface protein expression and NKactivating ligand expression. In one embodiment, a cell derived from ahuman pluripotent stem cell, such as a pancreatic cell, is provided thatlack some or all classic HLA-Class I cell surface protein expression andNK activating ligand expression. In one embodiment, there is provided amethod of preventing cell graft rejection by providing transplantedpancreatic cells wherein at least one MHC gene, such asbeta-2-microgobulin (B2M), and at least one NK activating ligand gene,such as Intercellular Adhesion Molecule 1 (ICAM-1), has been disrupted,deleted, modified, or inhibited. In another embodiment, there isprovided a method of preventing cell graft rejection by providingtransplanted pancreatic cells wherein the expression of at least one MHCprotein such as B2M and at least one NK activating ligand protein suchas ICAM-1 has been disrupted, deleted, modified, or inhibited.Disruption, deletion, modification, or inhibition of B2M, results indeficiency in all of HLA class I surface expression and function.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a reproduction from Haynes et al., supra, (which isincorporated herein in its entirety) showing different scenarios (A ofNK mediated response to target cells. In the absence of MHC-Class I andabsence of NK activating ligands on the target cell, inhibitory andactivating receptors on NK cells are not engaged and NK cells remainunresponsive (Scenario A). In the presence of MHC Class I, but in theabsence of NK activating ligands on the target cell, inhibitoryreceptors on NK cells are engaged but activating receptors on NK cellsare not engaged and NK cells remain unresponsive (Scenario B). In theabsence of self-MHC-Class I but in the presence of NK activating ligandson the target cell, inhibitory receptors on NK cells are not engaged butactivating receptors on NK cells are engaged and NK cells attack(Scenario C). In the presence of MHC-Class I and NK activating ligandson the target cell, inhibitory and activating receptors on NK cells areengaged and the outcome is determined by a balance of signals (ScenarioD).

FIG. 2A shows representative flow cytometry analysis of B2M cell surfaceprotein expression on wild-type (WT) hES cells without IFN-γ (Line B)and after exposure to IFN-γ (Line A). The shaded region is backgroundexpression with no antibody staining. Exposure to IFN-γ increases B2Mcell surface protein expression in WT hES cells.

FIG. 2B shows representative flow cytometry analysis of B2M cell surfaceprotein expression on B2M knockout (B2M −/−) hES cells without IFN-γ(Line B) and after exposure to IFN-γ (Line A). B2M −/− hES cells havevery little B2M cell surface protein expression which does notsignificantly change after exposure to IFN-γ.

FIG. 3A shows representative flow cytometry analysis of HLA-ABC cellsurface protein expression on WT hES cells without IFN-γ (Line B) andafter exposure to IFN-γ (Line A) using pan HLA Class I antibody. Theshaded region is background expression with no antibody staining.

FIG. 3B shows representative flow cytometry analysis of HLA-ABC cellsurface protein expression on B2M knockout hES cells without IFN-γ (LineB) and after exposure to IFN-γ (Line A). B2M −/− hES cells have nodetectable HLA-ABC cell surface protein expression.

FIG. 4A shows representative flow cytometry analysis of B2M cell surfaceprotein expression on WT pancreatic endoderm cells (PEC) without IFN-γ(Line B) and after exposure to IFN-γ (Line A). The shaded region isbackground expression with no antibody staining. Exposure to IFN-γincreases B2M cell surface protein expression in WT PEC.

FIG. 4B shows representative flow cytometry analysis of B2M cell surfaceprotein expression on B2M knockout PEC without IFN-γ (Line B) and afterexposure to IFN-γ (Line A). B2M −/− PEC have no detectable B2M cellsurface protein expression.

FIG. 5A shows representative flow cytometry analysis of HLA-ABC cellsurface protein expression on WT PEC without IFN-γ (Line B) and afterexposure to IFN-γ (Line A). The shaded region is background expressionwith no antibody staining. Exposure to IFN-γ increases HLA-ABC cellsurface protein expression in WT PEC.

FIG. 5B shows representative flow cytometry analysis of HLA-ABC cellsurface protein expression on B2M knockout PEC without IFN-γ (Line B)and after exposure to IFN-γ (Line A). B2M −/− PEC have no detectableHLA-ABC cell surface protein expression.

FIG. 6A shows representative flow cytometry analysis of ICAM-1 cellsurface protein expression on WT hES cells without IFN-γ (Line B) andafter exposure to IFN-γ (Line A). The shaded region is backgroundexpression with no antibody staining. Exposure to IFN-γ increases ICAM-1cell surface protein expression in WT hES cells.

FIG. 6B shows representative flow cytometry analysis of ICAM-1 cellsurface protein expression on B2M knockout hES cells without IFN-γ (LineB) and after exposure to IFN-γ (Line A). B2M −/− hES cells have similarICAM-1 cell surface protein expression as WT hES cells, before and afterIFN-γ exposure.

FIG. 7A shows representative flow cytometry analysis of ICAM-1 cellsurface protein expression on WT PEC without IFN-γ (Line B) and afterexposure to IFN-γ (Line A). The shaded region is background expressionwith no antibody staining. Exposure to IFN-γ increases ICAM-1 cellsurface protein expression in WT PEC.

FIG. 7B shows representative flow cytometry analysis of ICAM-1 cellsurface protein expression on B2M knockout PEC without IFN-γ (Line B)and after exposure to IFN-γ (Line A). After exposure to IFN-γ, the B2M−/− PEC have similar ICAM-1 cell surface protein expression as WT PECwhich is greater than that of the background (shaded region).

FIG. 8 is a bar graph showing mRNA expression data (Affymetrixexpression array) for ICAM-1 in WT hES cells, B2M (−/−) hES cells, WTPEC, and B2M (−/−) PEC each not exposed to IFN-γ (control) or exposed toIFN-γ. ICAM-1 expression is also assessed in cells known to have lowICAM cell surface protein expression: cancer cells (K562 and SKBR3),transplanted PEC that was allowed to mature to insulin producing cellsin vivo, human islet cells and two different samples of peripheral bloodmononuclear cells (PBMC) (no exposure to IFN-γ). ICAM-1 mRNA expressionis increased after exposure of hES cells (WT or B2M−/−) or PEC (WT orB2M−/−) to IFN-γ.

FIG. 9 shows representative flow cytometry analysis of CD58 (alias:LFA-3) cell surface protein expression on WT PEC without exposure toIFN-γ (Line B) and after exposure to IFN-γ (Line A). Line C isbackground expression with no antibody staining. Exposure to IFN-γ onlyslightly increases CD58 cell surface protein expression in WT PECcompared to untreated control. Antibody from BioLegend, Cat#330909.

FIG. 10 shows representative flow cytometry analysis of CD155 cellsurface protein expression on WT PEC without exposure to IFN-γ (Line B)and after exposure to IFN-γ (Line A). Line C is background expressionwith no antibody staining. After exposure to IFN-γ, the WT PEC havesimilar CD155 cell surface protein expression as WT untreated PECcontrol. Gene symbol PVR (aliases: CD155, NECL-5, HVED). Antibody fromMilteneyi Biotech Inc., Cat. #130-105-905.

FIG. 11 shows representative flow cytometry analysis of CEACAM1(aliases: CD66a, BGP, BGP1) cell surface protein expression on WT PECwithout exposure to IFN-γ (Line B) and after exposure to IFN-γ (Line A).Line C is background expression with no antibody staining. Exposure toIFN-γ slightly increases CEACAM1 cell surface protein expression in WTPEC compared to untreated control. Antibody from Milteneyi Biotech Inc.,Cat. #130-098-858.

FIG. 12 shows representative flow cytometry analysis of BAT3 cellsurface protein expression on WT PEC without exposure to IFN-γ (Line B)and after exposure to IFN-γ (Line A). Line C is background expressionwith no antibody staining. WT PEC in untreated control have similar BAT3cell surface protein expression as WT PEC exposed to IFN-γ. Gene symbolBAG6 (aliases: BAT3, HLA-B-associated transcript 3). Antibody fromAbcam, Inc., Cat. ab210838.

FIG. 13 shows representative flow cytometry analysis of CADM1 (aliases:NECL2, TSLC1, IGSF4, RA175) cell surface protein expression on WT PECwithout exposure to IFN-γ (Line B) and after exposure to IFN-γ (Line A).Line C is background expression with no antibody staining. Afterexposure to IFN-γ, WT PEC have similar CADM1 cell surface proteinexpression as untreated control. Antibody from MBL International Corp.Cat. #CM004-4.

FIG. 14 shows representative flow cytometry analysis of CD112 cellsurface protein expression on WT PEC without exposure to IFN-γ (Line B)and after exposure to IFN-γ (Line A). Line C is background expressionwith no antibody staining. After exposure to IFN-γ, the WT PEC havesimilar CD112 cell surface protein expression as untreated control. Genesymbol PVRL2 (aliases; CD112, Nectin-2, PVRR2, HVEB). Antibody fromMilteneyi Biotech Inc., Cat. #130-109-056.

FIG. 15 is a bar graph showing a reduction in NK cell lysis of targetcells after blocking ICAM-1 expression in the target cell with ananti-ICAM1 antibody (left to right, WT hESC, WT hESC exposed to IFN-γ,B2M −/−hESC, B2M −/−hES exposed to IFN-γ, WT PEC, WT PEC exposed toIFN-γ, B2M −/− PEC, B2M −/− PEC exposed to IFN-γ, K562-control cell linefor the NK cytotoxicity assay).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [9511-99296-03 Sequence Listing, May 3, 2019, 1.58 KB] which isincorporated by reference herein.

DETAILED DESCRIPTION

MHC-Class I molecules are one of two primary classes of majorhistocompatibility complex (MHC) molecules (the other being MHC-ClassII). Their function is to display peptide fragments of non-self proteinsfrom within the cell to cytotoxic T cells; this will trigger animmediate response from the immune system against a particular non-selfantigen displayed with the help of an MHC-Class I protein.

In humans, the HLAs corresponding to MHC-Class I are HLA-A, HLA-B,HLA-C, HLA-E, HLA-F, and HLA-G. The human HLA-E, HLA-F, and HLA-G arenon-classical MHC class I molecules characterized by limitedpolymorphism and a lower cell surface expression than the classicalparalogues (HLA-A, -B and -C). All MHC class I proteins must associatewith β2-microglobulin (B2M) to produce a functional heterodimer MHCClass I protein complex prior to functional expression on the cellsurface. MHC-Class I molecules can also serve as an inhibitory ligandfor NK cells. Reduction in the normal levels of cell surface MHC-ClassI, activates NK cell killing.

Historically, it was believed that target cells bearing MHC-Class Iinhibitory ligands evade attack when exposed to NK cells because of theassumed dominate nature of the MHC-Class I's inhibitory signal (see FIG.1 scenario B reproduced from Harrison's Principles of Internal Medicine19 E (Vol. 1 and Vol. 2) A Major Histocompatibility Complex, Part 15,FIG. 372e-4 therein). But, Applicants surprisingly found the opposite tobe true.

It has been shown that exposure of cells to IFN-γ increases mRNAexpression of MHC-Class I molecules and also MHC Class I protein complexexpression on the cell surface. It is expected that this increase inMHC-Class I expression inhibits NK cells. Applicants discovered thatwild type (WT) hES cells when exposed to IFN-γ (which has been shown toincrease MHC-Class I molecules on the cell surface of hES cells, FIGS.2A and 3A) have increased NK cell-mediated cytotoxicity. See FIG. 15showing NK cell mediated toxicity (lysis) increases from 58% to 79%(compare first bars in condition 1 and 2 in FIG. 15). The same was truewhen WT PEC cells were exposed to IFN-γ, the cells also had increasedB2M and HLA-ABC expression, see FIGS. 4A and 5A and NK mediated toxicity(lysis) increased from 33% to 53% (compare first bars in conditions 5and 6 in FIG. 15). This data suggested that the key to overcoming ahost's NK cell immune response is not in overexpressing inhibitoryMHC-Class I signals but in blocking NK activating ligand signals.

To further test this hypothesis in the context of accentuated NKcell-mediated cytotoxicity, Applicants made a B2M−/− (knockout) hES cell(similar to FIG. 1 scenario C). As expected, B2M−/− eliminated cellsurface expression of MHC Class I molecules on hES cells (FIGS. 2B and3B) and PEC (FIGS. 4B and 5B). Also as expected, the B2M−/− cellsexhibited increased NK cell-mediated lysis relative to the WT cells, 58%to 71% for hES cells and 33% to 54% for PEC (compare first bars incondition 1 vs. 3 or 5 vs. 7 in FIG. 15). Applicants discovered thatexposure of B2M−/− hES cells or PEC to IFN-γ further increased thepercentage of NK cell mediated toxicity (lysis) (compare first bars incondition 2 vs. 4 and 6 vs. 8 in FIG. 15). Correspondingly, Applicantsdiscovered that NK cell activating ligand cell surface expression (FIGS.6B and 7B), and mRNA expression (FIG. 8) is increased under exposure toIFN-γ. This data suggested that NK cell activating ligands on the targetcell play a critical role in the cytotoxicity of NK cells and led to thehypothesis that inhibiting NK cell activating ligand expression couldprotect against NK cell mediated cytotoxicity in the context of reducedMCH Class I expression, for example in the context of B2M−/−.

To determine whether NK cell toxicity may be reduced by inhibiting theeffect of the NK activating ligands on target cells, Applicants blockedthe expression of NK activating ligand in WT and B2M −/− hES cells andPEC, for example, using an ICAM1 blocking antibody to block ICAM1protein on the target cell surface. The Applicants surprisinglydiscovered that cell lysis of target cells was reduced (compare firstbars to 2nd and third bars for conditions 2, 4, 6 and 8 in FIG. 15).Thus, Applicants discovered that cell lysis by NK cells can be reducedby blocking an NK activating ligand. Blocking ICAM1 expression using anantibody against an NK activating ligand in B2M−/− cells is the proof ofconcept for producing a cell having a double knockout (HLA-Class I geneknockout and NK activating ligand gene knockout). In doing so,Applicants can transition target cells (e.g. hES and/or pancreaticlineage cells) from scenario C to A in FIG. 1. Specifically, the cells,tissues and organs of the invention have inhibited or no HLA-Class Icell surface protein expression (B2M −/−) and inhibited or no NKactivating ligand cell surface protein expression (e.g., ICAM1 −/−).Inhibiting cell surface protein expression can be achieved by knockingout the gene or blocking expression of the protein using an antibody.Other strategies for interfering with cell surface protein expressioninclude using anti-sense RNA, RNA decoys, ribozymes, RNA aptamers,siRNA, shRNA/miRNA, Transdominant negative proteins (TNPs),chimeric/fusion proteins, Nucleases, Chemokine ligands, Anti-infectiouscellular proteins, Intracellular antibodies (sFvs), Nucleoside analogues(NRTIs), non-nucleoside analogues (NNRTIs), Integrase inhibitors(Oligonucleotides, dinucleotides and chemical agents), and proteaseinhibitors. A double or multiple gene knockout would effectively preventboth cytotoxic T cell (CTL) mediated and NK cell mediated toxicitybecause there would be little to no HLA-Class I and little to no NKactivating ligand proteins expressed on the cell surface for the CTL orNK cell to bind to. Further, in order to completely eliminate NKactivation, Applicants anticipate that expression of multiple NKactivating ligands will need to be eliminated/reduced either by geneknockout in the target cell (e.g. the hES cell-derived cell therapy), orby using a blocking antibody or other strategies now known or developedin the future.

NK Cell Activating Ligand Blocking Agents

According to one aspect of the invention, a method of treatment tosuppress NK cell function is provided. According to another aspect ofthe invention, a method of treatment to suppress at least one immuneresponse is provided. Each method involves administering to a subject inneed of treatment an agent that inhibits NK cell function. In someembodiments, the agent is an antibody. In some embodiments the antibodyselectively binds to a NK cell activating ligand on a target cell.

It is contemplated that reagents of various types, including antibodiesand blocking proteins can be used to interfere with adhesion between NKcells and target cells' NK activating ligands.

In certain embodiments, such NK activating ligands are selected fromTable 1.

TABLE 1 Natural Killer (NK) Activating Ligands Category GENE IDDescription Category 1 ICAM1 Intercellular adhesion molecule 1 Known NKCEACAM1 Carcinoembryonic antigen-related cell activating adhesionmolecule 1 ligands BAG6 Large proline-rich protein BAG6 CADM1 Celladhesion molecule 1 CD58 Lymphocyte function-associated antigen 3 CD72B-cell differentiation antigen CD72 CD74 HLA-E MICA MHC-Class Ipolypeptide-related sequence A MICB MHC-Class I polypeptide-relatedsequence B PVR Poliovirus receptor PVRL2 NECTIN2 Category 2 BTN3A3Butyrophilin subfamily 3 member A3 Potential NK CD47 Leukocyte surfaceantigen CD47 activating CTSS Cathepsin S ligands NTRK2 BDNF/NT-3 growthfactors receptor identified RTP4 Receptor-transporting protein 4 fromRNA TLR3/CD283 Toll-like receptor 3 expression TMEM140 Transmembraneprotein 140 array data TMPRSS3 Transmembrane protease serine 3(upregulated BST2/CD317 Bone marrow stromal antigen 2 in PEC and/ BTN3A1Butyrophilin subfamily 3 member A1 or ESC after CD40 IFNγ) EPSTI1Epithelial-stromal interaction protein 1 ERAP1 Endoplasmic reticulumaminopeptidase 1 ERAP2 Endoplasmic reticulum aminopeptidase 2 GJD3 Gapjunction delta-3 protein HCP5 HLA-Class I histocompatibility antigenprotein P5 IFI6 Interferon alpha-inducible protein 6 IFITM1Interferon-induced transmembrane protein 1 IFITM2 Interferon-inducedtransmembrane protein 2 IFITM3 Interferon-induced transmembrane protein3 LGALS3BP Galectin-3-binding protein Category 3 C1QBP Complementcomponent 1 Q subcomponent-binding protein, mitochondrial Potential NKCD24 activating CD55 Complement decay-accelerating factor ligands CD9Leukocyte antigen MIC3 identified GJA1 Gap junction alpha-1 protein fromRNA GPRC5B G-protein coupled receptor family C group expression 5 memberB array data HMMR Hyaluronan mediated motility receptor (upregulatedICAM3 Intercellular adhesion molecule 3 in ESC) IGSF5 Immunoglobulinsuperfamily member 5 SYNGR3 Synaptogyrin-3 TFRC/CD71 Transferrinreceptor protein 1 THY-1/CD90 Thy-1 membrane glycoprotein TMEM68Transmembrane protein 68 TMEM97 Transmembrane protein 97 ANKRD27 Ankyrinrepeat domain-containing protein 27

NK activating ligands are further described in Pegram et al., Activatingand inhibitory receptors of NK cells Immunology and Cell Biology (2011)89, 216-224 which is herein incorporated by reference in its entirety.

Hypoimmunogenic hES Cells and Cells Derived Therefrom

HLA is a cell surface molecule that is encoded by a large gene familyand can be divided into class I and class II molecules. HLA-Class Imolecules are found on the surface of every nucleated cell and is thefocus of the invention described herein. HLA mismatch between donor(target) cells and the recipient's immune cells (e.g. T cells) duringtransplantation often results in immune rejection or graft rejection.HLA-Class I complexes structurally consist of a polymorphic heavy chainconsisting of HLA-Class I peptides (e.g., HLA-A, HLA-B and HLA-C) and alight chain beta-2-microglobulin (β2m or B2M). In the absence of B2M,class I HLAs cannot be properly assembled and are also not expressed onthe cell surface or cell membrane. In the invention described herein,Applicants produced hES cell lines and cells derived therefrom bydisrupting (a few base pairs are added or removed, creating a frameshift in the mRNA/protein and a loss of function mutation) the B2M gene,and thereby depleting HLA-Class 1 expression from the cell surface inhESCs.

The above methodology can also be used to produce or generate hES celllines and cells derived therefrom by additionally disrupting genes thatencode for NK activating ligands, such as ICAM1. Thus, in one embodimentof the invention, compositions and methods are provided to make a targetcell that is missing at least one HLA-Class I antigen and at least oneNK activating ligand, and thereby creating a hypoimmunogenic cell. Sucha hypoimmunogenic cell is expected to be less prone to immune rejectionby a subject into whom such cells are transplanted. When transplanted,this hypoimmunogenic cell should engraft (not be rejected). In oneembodiment, such a target cell is capable of engrafting and survivingwith little to no immune suppression required of the recipient.

In one embodiment, the inhibition, reduction, and/or deletion of bothHLA-Class I expression and NK activating ligand expression (or HLA-ClassI deficient and NK activating ligand deficient) in hESC cells and cellsderived therefrom can serve as a universal donor cell source fortransplantation therapy. These double knockouts (HLA-Class I deficientand NK activating ligand deficient) can be transplanted universallywithout minor histocompatibility complex (MiHC) matching, humanleukocyte antigen (HLA) matching or immune suppression.

Disclosed herein are novel in vitro derived hypoimmunogenic compositionsand cells. Specifically, in certain embodiments, the inventionsdisclosed herein relate to a stem cell, the genome of which has beenaltered (modified) to reduce or delete critical components of both aMHC-Class I gene(s) and a NK activating ligand gene(s). In certainembodiments, the inventions disclosed herein relate to pancreaticlineage cells such as pancreatic endoderm cells, pancreatic epithelialcells, pancreatic progenitor cells, pancreatic precursor endocrinecells, pancreatic endocrine cells, pancreatic pre-beta cells, orpancreatic beta cells, the genome of which has been altered (modified)to reduce or delete critical components of both a MHC-Class I gene(s)and a NK activating ligand gene(s) thereby generating hypoimmunogenicpancreatic-lineage type cells. Natural killer activating ligands includebut are not limited to the ligands listed in Table 1, from category 1,2, 3, or combinations thereof. Natural killer activating ligandsinclude, for example ICAM-1, CEACAM1, CADM1, MICA and MICB. MHC-Class Igenes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G and B2M. Incertain aspects, such reduced expression or knock out of the MHC-Class Iand/or MHC-Class II genes is accomplished by directly and/or indirectlytargeting the NLRCS, B2M and CIITA genes and other components of the MHCenhanceosome (an enhanceosome is a higher-order protein complexassembled at the enhancer and regulates expression of a target gene,e.g., transcriptional regulators of MHC-Class I or MHC-Class II).

Also disclosed herein are methods of preparing hypoimmunogenic cells,the method comprising modulating expression of one or more NK activatingligands expressed by the cell and modulating expression of one or moreMHC-Class I and/or MHC-Class II by the cell, thereby preparing thehypoimmunogenic cell. In certain aspects, modulating cell surfaceprotein expression of one or more MHC-Class I and/or MHC-Class IIcomplexes comprises reducing, inhibiting and/or interfering with theexpression of one or more MHC-Class I and/or MHC-Class II genes orproteins. In certain embodiments, modulating expression of the one ormore MHC-Class I and/or MHC-Class II complexes comprises deleting one ormore genes encoding one or more transcriptional regulators of MHC-ClassI or MHC-Class II from at least one allele of the cell. For example, incertain embodiments such methods comprise deleting one or more genesencoding one or more of the transcriptional regulators of MHC-Class I orMHC-Class II genes selected from the group consisting of LRCS, CIITA,B2M and combinations thereof. In certain aspects, modulating expressionof the one or more NK activating ligands comprises deleting, inhibiting,or reducing expression of one or more genes encoding a NK activatingligand. In certain embodiments, such NK activating ligands are selectedfrom Table 1. In certain embodiments, such NK activating ligands areselected from Table 1, category 1, 2, 3 or combinations thereof. Incertain embodiments, such NK activating ligands are selected from Table1 category 1, 2 and 3. In certain embodiments, such NK activatingligands are selected from Table 1 category 1 and 3. In certainembodiments, such NK activating ligands are selected from Table 1category 1 and 2. In certain embodiments, such NK activating ligands areselected from Table 1 category 2 and 3. In certain embodiments, such NKactivating ligands are selected from the group consisting of ICAM-1,CEACAM1, CADM1 MICA, MICB and combinations thereof.

In certain embodiments, the implanted hypoimmunogenic cells are in amedia free of animal-sourced products, e.g. xenofree products.

The present invention contemplates altering target polynucleotidesequences in any manner which is available to the skilled artisan, forexample, utilizing any of zinc-finger nucleases (ZFN or ZNF), TALEN or aCRISPR/Cas systems or traditional homologous recombination techniques.Such CRISPR/Cas systems can employ a variety of Cas proteins (Haft etal. PLoS Comput Biol. 2005; 1(6)e60). In some embodiments, theCRISPR/Cas system is a CRISPR type I system. In some embodiments, theCRISPR/Cas system is a CRISPR type II system. In some embodiments, theCRISPR/Cas system is a CRISPR type V system. NEXTGEN™ CRISPR(Transposagen Inc., Lexington Ky.), which incorporates dual guide RNA'sand a catalytically inactive Cas9 protein fused to the FokI nuclease canalso be used to alter a target polynucleotide sequence. Other methods oftargeting polynucleotide sequences to reduce or ablate expression intarget cells now known to the skilled artisan or later discovered can beutilized to generate the hypoimmunogenic cells described herein.

In some embodiments, the alteration results in reduced expression of thetarget polynucleotide sequence. In some embodiments, the alteration is ahomozygous alteration. In some embodiments, the alteration is aheterozygous alteration.

In some embodiments, the target polynucleotide sequence is a genomicsequence. In some embodiments, the target polynucleotide sequence is ahuman genomic sequence. In some embodiments, the target polynucleotidesequence is a mammalian genomic sequence. In some embodiments, thetarget polynucleotide sequence is a vertebrate genomic sequence.

In some embodiments, the hypoimmunogenic cells are embryonic stem cells.In certain embodiments, the hypoimmunogenic cells are pluripotent stemcells. In certain embodiments, the hypoimmunogenic cells are inducedpluripotent stem cells, reprogrammed cells, dedifferentiated ortransdifferentiated cells. In certain embodiments, the hypoimmunogeniccells are multipotent pancreatic progenitor cells. In certainembodiments, the hypoimmunogenic cells are singly hormonal orpolyhormonal cells. In certain embodiments, the hypoimmunogenic cellsare mesendoderm cells, definitive endoderm cells, PDX1-negative foregutendoderm cells, PDX1-positive foregut endoderm cells, pancreaticendoderm cells, endocrine progenitor/precursor cells, endocrine cells,properly specified endocrine cells, immature endocrine cells, orfunctional beta-cells. In some embodiments, the hypoimmunogenic cellscan be homogenous or heterogeneous cell populations. In someembodiments, the hypoimmunogenic cell are cells producing one or morebiologically active substances of interest. Hypoimmunogenic cells maynot initially be therapeutically active when first implanted, e.g.pancreatic progenitors or PDX1-positive pancreatic endoderm, but oncetransplanted they further develop and mature and have a therapeuticeffect.

In some embodiments, the hypoimmunogenic cells may be any cell capableof being derived from human pluripotent stem cells including but notlimited to any cell, tissue, or organ and can include skin cells, betacells (i.e., cells in the pancreas located in the islets of Langerhans),parathyroid cells, intestinal cells, endocrine cells cardiac cells,brain cells, kidney cells, liver cells, cells of the digestive tract andaccessory digestive organs, salivary gland cells, adrenal gland cells,prostate cells, lung cells, pancreatic cells, bone cells, immune cells,hematopoietic cells, vascular cells, cells of the eye, connective tissuecells, musculoskeletal cells, bone tissue, musculoskeletal tissue,cornea tissue, skin tissue, heart valves, blood vessels, immune cells,connective tissue, lung tissue, skin, a cornea, a kidney, a liver, alung, a pancreas, a heart, and intestine.

In some embodiments, the hypoimmunogenic cell can be individual (single)cells in suspension or cell aggregates. In some embodiments, thehypoimmunogenic cells include totipotent cells. In one embodiment, thehypoimmunogenic cells include multipotent cells. In one embodiment, thehypoimmunogenic cells include unipotent cells.

In some embodiments, the hypoimmunogenic cells are derived from thepluripotent cell population lacking functional HLA-Class I expressionand NK activating ligand expression. The derived cells can be selectedfrom the group consisting of: any cell, tissue, or organ and can includeskin cells, beta cells (i.e., cells in the pancreas located in theislets of Langerhans), parathyroid cells, intestinal cells, endocrinecells cardiac cells, brain cells, kidney cells, liver cells, cells ofthe digestive tract and accessory digestive organs, salivary glandcells, adrenal gland cells, prostate cells, lung cells, pancreaticcells, bone cells, immune cells, hematopoietic cells, vascular cells,cells of the eye, connective tissue cells, musculoskeletal cells, bonetissue, musculoskeletal tissue, cornea tissue, skin tissue, heartvalves, blood vessels, immune cells, connective tissue, lung tissue,skin, a cornea, a kidney, a liver, a lung, a pancreas, a heart, andintestine.

The hypoimmunogenic cell, tissue and/or organ to be transplanted can besyngeneic or allogenic to the subject receiving the transplant.

In one embodiment the hypoimmunogenic cell is a human pluripotent cell.In one embodiment the hypoimmunogenic cell is a human pancreatic-lineagecell. In one embodiment the hypoimmunogenic cell is a human pancreaticendoderm cell. In one embodiment the hypoimmunogenic cell is a humanpancreatic precursor cell. In one embodiment the hypoimmunogenic cell isa human pancreatic progenitor cell. In one embodiment thehypoimmunogenic cell is a human pancreatic endocrine cell. In oneembodiment the hypoimmunogenic cell is a human pancreatic endocrineprecursor cell. In one embodiment the hypoimmunogenic cell is a humanpancreatic endocrine pre-beta cell. In one embodiment thehypoimmunogenic cell is a human pancreatic beta cell. In one embodimentthe hypoimmunogenic cell is a human pancreatic singly hormonal orpolyhormonal cell. In one embodiment the hypoimmunogenic cell is a humaninsulin expressing cell.

In one embodiment, the hypoimmunogenic cells are well known, publiclyavailable pluripotent cell lines. The invention described herein isuseful with all hES cell and iPSC lines, and at least hESC, e.g., CyT49,CyT25, CyT203 and CyT212. Pluripotent cell lines include those cellsavailable for commercial purchase from WiCell on the world wide web atwicell.org/home/stem-cell-lines/order-stem-cell-lines/obtain-stem-cell-lines.cmsxand specifically include BG01, BG02, and BG03.

In one embodiment, the hypoimmunogenic cells are substantially similarto the cells described in D'Amour et al. “Production of PancreaticHormone-Expressing Endocrine Cells From Human Embryonic Stem Cells”(Nov. 1, 2006) Nature Biotechnology 24, 1392-1401 which is hereinincorporated by reference in its entirety. D'Amour et al. describe a 5step differentiation protocol: stage 1 (results in mostly definitiveendoderm production), stage 2 (results in mostly PDX1-negative foregutendoderm production), stage 3 (results in mostly PDX1-positive foregutendoderm production), stage 4 (results in mostly pancreatic endodermalso called multipotent pancreatic progenitor or pancreatic endocrineprogenitor production) and stage 5 (results in mostly hormone-expressingendocrine cell production). In one embodiment, the hypoimmunogenic cellsare substantially similar to that described in U.S. Pat. Nos. 7,510,876,7,695,965, 7,985,585, 8,586,357, 8,633,024 and 8,129,182 (which areherein incorporated by reference in their entirety).

In one embodiment, the hypoimmunogenic cells are substantially similarto the cells described in Schulz et al. A Scalable System for Productionof Functional Pancreatic Progenitors from Human Embryonic Stem Cells,PLoS One 7:5 1-17 (2012) which is herein incorporated in its entirety byreference. Schulz et. al. describe hESC expansion and banking methodsand a suspension-based differentiation system. Specifically,undifferentiated pluripotent cells were aggregated into clusters indynamic rotational suspension culture, followed by differentiation enmasse for two weeks with a four-stage protocol. Briefly, from hES cellaggregate suspensions, hESC monolayers are dissociated with Accutase(Innovative Cell Technologies), collected and resuspended at 1×106cells/mL in StemPro hESC SFM (Life Technologies; combined DMEM/F12containing Glutamax, StemPro hESC supplement, BSA, and 1% (v/v)Penicillin/streptomycin; omitted FGF-2 and 2-Mercaptoethanol). Thesingle cell suspensions were dispensed to non-TC treated 6-well plates(5.5 mL/well) and rotated at 95 rpm on an Innova 2000 rotator (NewBrunswick Scientific), or dispensed to 500 mL Nalgene filter receiverstorage bottles (150 mL/bottle) and rotated at 65 rpm on a SartoriusCertomat RM-50 rotator (configured with a 5 cm axis of rotation). Cellswere rotated overnight in a 37° C./8% CO2 incubator and formedaggregates of approximately 100-200m. For aggregate diameters between100-200 μm rotation speeds between 60-140 rpm for a 6-well dish can beused; rotation speeds between 5-20 rpm for a 500 mL bottle can be used.Differentiation of suspension aggregates involved only a fewmodifications from D'Amour. The TGF-βRI kinase Inhibitor IV was includedduring Stage-2, and retinoic acid was replaced with a more stableretinoid analog, TTNPB (3 nM), during Stage-3. The growth factors KGF(50 ng/mL) and EGF (50 ng/mL) were added to Stage-4 to preserve cellmass. Noggin (50 ng/mL) was also included at Stage-4. In one embodiment,the hypoimmunogenic cells are substantially similar to that described inU.S. Pat. Nos. 8,008,075 and 8,895,300 (which are herein incorporated byreference in their entirety).

In one embodiment, hypoimmunogenic cells are substantially similar tothe cells described in Agulnick et al. Insulin-Producing Endocrine CellsDifferentiated In Vitro From Human Embryonic Stem Cells Function inMacroencapsulation Devices In Vivo Stem Cells Translationalmedicine4:1-9 (2015) which is herein incorporated in its entirety by reference.Agulnick et al. described a modified protocol for making pancreaticprogenitors cells such that 73%-80% of the cell population consist ofPDX1-positive (PDX1+) and NKX6.1+ pancreatic progenitors. The pancreaticprogenitor cells were further differentiated into islet-like cells (ICs)that reproducibly contained 73%-89% endocrine cells, of whichapproximately 40%-50% expressed insulin. A large fraction of theseinsulin-positive cells were single hormone-positive and expressed thetranscription factors PDX1 and NKX6.1. Agulnick et al. describe aprotocol wherein the Schulz et al. 2012 protocol was modified byadditionally treating with activin A, Wnt3A, and heregulin β1 at stage 3(days 5-7) and with activin A and heregulin β1 at stage 4 (days 7-13).In one embodiment, the hypoimmunogenic cells are substantially similarto the cells described in U.S. Pat. No. 8,859,286 (which is hereinincorporated by reference in its entirety).

Growth, passaging and proliferation of human embryonic stem cells can beperformed substantially as described in U.S. Pat. Nos. 7,964,402;8,211,699; 8,334,138; 8,008,07; and 8,153,429.

Standard Manufacturing Protocol

A standard manufacturing method for making pancreatic endoderm cells(PEC) derived from hESC is disclosed below in Table 2.

Roller 6-well Time Bottle tray point Stage Speed Speed (day) (1-4) MediaCondition (rpm) (rpm) d (−1) hESC XF HA; SP 31 95 Agg. d 0 1r0.2FBS-ITS1: 5000 A100 W50 31 95 d 1 r0.2FBS-ITS1: 5000 A100 31 95 d 22 r0.2FBS-ITS1: 1000 K25 IV 31 95 d 3 r0.2FBS-ITS1: 1000 K25 31 95 d 4r0.2FBS-ITS1: 1000 K25 31 105 d 5 3 db-CTT3 N50 31 105 d 6 db-CTT3 N5031 105 d 7 db-CTT3 N50 31 105 d 8 4 db-N50 K50 E50 31 105 d 9 db-N50 K50E50 31 95 d 10 db-N50 K50 E50 31 95 d 11 db-N50 K50 E50 31 95 d 12db-N50 K50 E50 31 95

hESC Agg.: hESC aggregates; XF HA: DMEM/F12 containing GlutaMAX,supplemented with 10% v/v of Xeno-free KnockOut Serum Replacement, 1%v/v non-essential amino acids, 1% v/v penicillin/streptomycin (all fromLife Technologies), 10 ng/mL heregulin-1β (Peprotech) and 10 ng/mLactivin A (R&D Systems); SP: StemPro® hESC SFM (Life Technologies);r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1× GlutaMAX-1 (LifeTechnologies), 1% v/v penicillin/streptomycin; ITS:Insulin-Transferrin-Selenium (Life Technologies) diluted 1:5000 or1:1000; A100:100 ng/mL recombinant human Activin A (R&D Systems); W50:50ng/mL recombinant mouse Wnt3A (R&D Systems); K25:25 ng/mL recombinanthuman KGF (R&D Systems); IV: 2.5 μM TGF-β RI Kinase inhibitor IV (EMDBioscience); db: DMEM HI Glucose (HyClone) supplemented with 0.5×B-27Supplement (Life Technologies), 1× GlutaMAX, and 1% v/vpenicillin/streptomycin; CTT3: 0.25 μM KAAD-Cyclopamine (TorontoResearch Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50: 50 ng/mLrecombinant human Noggin (R&D Systems); K50: 50 ng/mL recombinant humanKGF (R&D Systems); E50: 50 ng/mL recombinant human EGF (R&D Systems).

Calcein Release Assay

Calcein release assay is a non-radioactive alternative for studying NKcell cytotoxicity. The target cells take up the fluorescent dye (calceinAM) and cytoplasmically convert it into the active fluorochrome, whichis only released from the cell upon lysis. Lysed cells release thefluorochrome into the supernatant, which is then harvested and theamount of fluorescence quantitated in a fluorometer. The percent celllysis is calculated from the amount of fluorescence present in thesupernatant after incubation in the presence or absence of NK cells(effectors), blocking antibody or both.

Specific lysis can be calculated by using the formula, %lysis=100×[(mean fluorescence with antibody−mean spontaneousfluorescence)/(mean maximum fluorescence−mean spontaneousfluorescence)]. Maximum fluorescence was determined by the lysis ofcells incubated with detergent (1% Triton X-100) and spontaneous lysiswas the fluorescence obtained with target cells without any antibody oreffector cells.

Various cell compositions derived from pluripotent stem cells andmethods thereof are described herein and can be found in Applicant'sU.S. patent application Ser. No. 10/486,408, entitled METHODS FORCULTURE OF HESC ON FEEDER CELLS, filed Aug. 6, 2002; Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004; Ser. No.11/115,868, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 26, 2005; Ser.No. 11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FORDIFFERENTIATING DEFINITIVE ENDODERM, filed Jun. 23, 2005; Ser. No.11/573,662, entitled METHODS FOR INCREASING DEFINITIVE ENDODERMDIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3KINASE INHIBITORS, filed Aug. 15, 2005; Ser. No. 12/729,084 entitledPDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed Oct. 27,2005; Ser. No. 12/093,590, entitled MARKERS OF DEFINITIVE ENDODERM,filed Nov. 14, 2005; Ser. No. 11/993,399, entitled EMBRYONIC STEM CELLCULTURE COMPOSITIONS AND METHODS OF USE THEREOF, filed Jun. 20, 2006;Ser. No. 11/588,693, entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUTENDODERM, filed Oct. 27, 2006; Ser. No. 11/681,687, entitled ENDOCRINEPROGENITOR/PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS ANDMETHODS OF PRODUCTION, filed Mar. 2, 2007; Ser. No. 11/807,223, entitledMETHODS FOR CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC,filed May 24, 2007; Ser. No. 11/773,944, entitled METHODS OF PRODUCINGPANCREATIC HORMONES, filed Jul. 5, 2007; Ser. No. 11/860,494, entitledMETHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION, filed Sep. 24,2007; Ser. No. 12/099,759, entitled METHODS OF PRODUCING PANCREATICHORMONES, filed Apr. 8, 2008; Ser. No. 12/107,020, entitled METHODS FORPURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMANEMBRYONIC STEM CELLS, filed Apr. 21, 2008; Ser. No. 12/618,659, entitledENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENTSTEM CELLS, filed Nov. 13, 2009; Ser. Nos. 12/765,714 and 13/761,078,both entitled CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMEDCELLS, filed Apr. 22, 2010 and Feb. 6, 2013; Ser. No. 11/838,054,entitled COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLECELLS, filed Aug. 13, 2007; Ser. No. 12/264,760, entitled STEM CELLAGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATIONTHEREOF, filed Nov. 4, 2008; Ser. No. 13/259,15, entitled SMALLMOLECULES SUPPORTING PLURIPOTENT CELL GROWTH, filed Apr. 27, 2010;PCT/US11/25628, entitled LOADING SYSTEM FOR AN ENCAPSULATION DEVICE,filed Feb. 21, 2011; Ser. No. 13/992,931, entitled AGENTS AND METHODSFOR INHIBITING PLURIPOTENT STEM CELLS, filed Dec. 28, 2010; and U.S.Design application Ser. No. 29/408,366 filed Dec. 12, 2011; Ser. No.29/408,368 filed Dec. 12, 2011; Ser. No. 29/423,365 filed May 31, 2012;and Ser. No. 29/447,944 filed Mar. 13, 2013; U.S. application Ser. No.14/201,630 entitled 3-DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATIONDEVICE ASSEMBLY, filed Mar. 7, 2014; and U.S. application Ser. No.14/106,330 entitled IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLSTO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS, filed Dec. 13,2013; PCT/US2016/061442, entitled PDX1 PANCREATIC ENDODERM CELLS IN CELLDELIVERY DEVICES AND METHODS THEREOF, filed Nov. 10, 2016; all of whichare herein incorporated by reference in their entirety.

Various cell compositions derived from pluripotent stem cells andmethods thereof are described herein and can be found in applicationsexclusively licensed by Applicant: U.S. Patent Publication no.2009/0269845 entitled Pluripotent cells filed Apr. 24, 2008; U.S. PatentPublication no. 2011/0014703 entitled Differentiation of Human EmbryonicStem Cells filed Jul. 20, 2010; U.S. Patent Publication no. 2011/0014702entitled Differentiation of Human Embryonic Stem Cells filed Jul. 19,2010; U.S. Patent Publication no. 2011/0151561 entitled Differentiationof Human Embryonic Stem Cells filed Dec. 16, 2010; U.S. PatentPublication no. 2010/0112692 entitled Differentiation of Human EmbryonicStem Cells filed Oct. 22, 2009; U.S. Patent Publication no. 2012/0052576entitled Differentiation of Pluripotent Stem Cells filed Aug. 17, 2011;U.S. Patent Publication no. 2010/0112693 entitled Differentiation ofhuman pluripotent stem cells filed Oct. 23, 2009; U.S. PatentPublication no. 2011/0151560 entitled Differentiation of human embryonicstem cells filed Dec. 16, 2010; U.S. Patent Publication no. 2010/0015100entitled Differentiation of human embryonic stem cells filed Jul. 31,2008; U.S. Patent Publication no. 2009/0170198 entitled Differentiationof human embryonic stem cells filed Nov. 25, 2008; U.S. PatentPublication no. 2015/0329828 entitled Use of Small Molecules to EnhanceMAFA Expression in Pancreatic Endocrine Cells filed May 7, 2015; U.S.Patent Publication no U.S. 2013/0330823 entitled Differentiation ofHuman Embryonic Stem Cells into Pancreatic Endocrine Cells filed Jun. 6,2013; International patent publication no. WO 2013/192005 entitledDifferentiation of human embryonic stem cells into pancreatic endocrinecells filed Jun. 13, 2013; U.S. Patent Publication no U.S. 2014/0242693entitled Suspension and clustering of human pluripotent stem cells fordifferentiation into pancreatic endocrine cells filed Dec. 30, 2013;U.S. Patent Publication no U.S. 2014/0295552 entitled Suspension andclustering of human pluripotent stem cells for differentiation intopancreatic endocrine cells filed Jun. 17, 2014; International patentpublication no. WO 2015/065524 entitled Suspension and clustering ofhuman pluripotent stem cells for differentiation into pancreaticendocrine cells filed May 21, 2014; U.S. Patent Publication no U.S.2013/0330823 entitled Differentiation of Human Embryonic Stem Cells intoPancreatic Endocrine Cells filed Jun. 6, 2013; U.S. Patent Publicationno U.S. 2014/0186953 entitled Differentiation of Human Embryonic StemCells Into Pancreatic Endocrine Cells Using HB9 Regulators filed Dec.18, 2013; U.S. application Ser. No. 14/963,730 filed December 9, 215;U.S. application Ser. No. 14/898,015 filed Dec. 11, 2015 all of whichare herein incorporated by reference in their entireties.

In one embodiment, hypoimmunogenic cells are encapsulated within a celldelivery device. The cell delivery device may comprise a non-wovenfabric. Cell delivery devices include various layers each of whichserves a function or multiple functions. In some embodiments, the celldelivery device includes both a cell-excluding membrane and a non-wovenfabric. In another embodiment, the delivery device is a TheraCyte(formerly Baxter) device (Irvine, Calif.). TheraCyte cell deliverydevices are further described in U.S. Pat. Nos. 6,773,458; 6,156,305;6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529;5,782,912; 5,741,330; 5,733,336; 5,713,888; 5,653,756; 5,593,440;5,569,462; 5,549,675; 5,545,223; 5,453,278; 5,421,923; 5,344,454;5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494, which are allherein incorporated by reference in their entireties.

In another embodiment, the delivery device is a device as substantiallydescribed in U.S. Pat. No. 8,278,106, and as described in U.S.application Ser. No. 14/201,630 filed Mar. 7, 2014 and in PCTApplication No. PCT/US2016/061442 filed Nov. 10, 2016, and in U.S.Design Ser. Nos. 29/447,944, 29/509,102, 29/484,363, 29/484,360,29/484,359, 29/484,357, 29/484,356, 29/484,355, 29/484,362, 29/484,358,29/408,366, 29/517,319, 29/408,368, 29/518,513, 29/518,516, 29/408,370,29/517,144, 29/423,365, 29/530,325, 29/584,046 which are all hereinincorporated by reference in their entireties. In other embodiments,cell delivery device or large capacity assembly consist of one or two ormore seals that further partition the lumen of the cell delivery device,i.e., a partition seal. See, e.g. Applicant's U.S. Design applicationSer. Nos. 29/408366, 29/408368, 29/408370, 29/423,365 and 29/584,046.

In one embodiment, hypoimmunogenic cells are implanted in a perforatedcell delivery device which provides direct cell-to-cell contact betweenhost vasculature and the encapsulated cells. Perforated means a hole orpore in the device. In some embodiments not all the layers of the deviceare perforated. For example see PCT Application No. PCT/US2016/0061442which is herein incorporated by reference in its entirety which discusesperforated cell delivery devices with perforations in just one layer,for example, the cell-excluding membrane; or, in just the cell-excludingmembrane and the non-woven fabric layer. In one embodiment,hypoimmunogenic cells are encapsulated in a perforated device surroundedby a non-woven fabric. In these embodiments, the non-woven fabric is onthe outside of the cell delivery device. Rather than affecting implantedcells, the non-woven fabric enhances host vascularization surroundingthe cell housing. See e.g. PCT/US2016/0061442 and U.S. Pat. No.8,278,106 (both of which are herein incorporated by reference in theirentirety) which describe perforated devices and device polymers.

In one embodiment, the holes/perforations are smaller than cellaggregates contained in the device, such as the hPSC-derived aggregates,e.g. definitive endoderm lineage cell aggregates, contained therein. Inone embodiment, a perforated cell delivery device implanted into a rator human contains perforations in just the cell-excluding membrane (theother layers of the device are not perforated) and wherein the holes areseparated by about 2 mm or more and wherein the hole diameter is lessthan about 100 microns is provided.

Hypoimmunogenic Cell Depletion (“Suicide Gene”)

The versatility of embryonic stem cells and induced pluripotent stem(iPS) cells to replace and restore any tissue in the body comes intandem with an increased risk of cancer. An increased cancer risk hasalso been associated with gene therapy. Hence, reprogrammed tissues,whether derived from ES cells or iPS cells (Takahashi, K. & Yamanaka, S.Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell 126, 663-676, (2006), andHanna, J. H., Saha, K. & Jaenisch, R. Pluripotency and CellularReprogramming: Facts, Hypotheses, Unresolved Issues. Cell 143, 508-525,(2010) both of which are herein incorporated by reference in theirentireties) or from other multipotent or progenitor cell, as well asfrom cells treated with gene therapy vectors, present safety concerns(Knoepfler, P. S. Deconstructing Stem Cell Tumorigenicity: A Roadmap toSafe Regenerative Medicine. Stem Cells 27, 1050-1056, (2009)incorporated by reference in its entirety). For example, subcutaneouslyimplanted iPS cells cause teratomas, and iPS chimeric animals developprimitive malignant cancers with high incidence (Takahashi, K. &Yamanaka, S. Induction of pluripotent stem cells from mouse embryonicand adult fibroblast cultures by defined factors. Cell 126, 663-676,(2006); Knoepfler, P. S. Deconstructing Stem Cell Tumorigenicity: ARoadmap to Safe Regenerative Medicine. Stem Cells 27, 1050-1056,(2009)). While benign teratomas may readily be removed by surgery,invasive cancers remain a risk with cell therapies.

Strategies for overcoming stem cell tumorigenicity, including a suicidegene strategy, have been considered (Knoepfler, P. S. DeconstructingStem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. StemCells 27, 1050-1056, (2009)). Specifically, a gene can be selectivelyintroduced into the implanted cell which encodes for an enzyme thatmetabolizes a systemically available pro-drug to an activeanti-neoplastic agent locally. For example, treatment with ganciclovir,which is converted by thymidine kinase into compounds that become toxicafter triphosphorylation by cellular kinases, resulted in destruction ofthe tumor cells in vitro. Thus, implanted cells can be modified toartificially generate exploitable biochemical differences between hosttissues and implanted cells. Targeting of the implanted cells isachieved by selection of the vector used to deliver the suicide gene, aswell as by the biology of suicide gene/prodrug system employed. As aresult, high doses of the drug generated only in the environment wherethe cells are implanted limits side effects in other tissues.

Hypoimmunogenic cell depletion may be accomplished by selectivelyintroducing a gene into the hypoimmunogenic cell, the expression ofwhich gene either directly results in hypoimmunogenic cell death orrenders the hypoimmunogenic cell specifically susceptible to otherpharmacological agents. In vivo or ex vivo depletion of hypoimmunogeniccell according to this method may be accomplished by delivering thedesired gene to the hypoimmunogenic cell using a viral gene deliverysystems such as, but not limited to a retrovirus, adenovirus or anadeno-associated virus gene delivery system. The desired viral deliverysystem may comprise a virus whose genome encodes a protein which, forexample, directly causes cell death, for example by inducing apoptosisof the hypoimmunogenic cell. Alternatively, the viral delivery systemmay contain a virus whose genome encodes, for example, a herpes simplexvirus thymidine kinase gene. Expression of the herpes simplex virusthymidine kinase gene in the hypoimmunogenic cell renders thehypoimmunogenic cell sensitive to pharmacologic doses of ganciclovir.Thus, subsequent contact of the virally transduced hypoimmunogenic cellwith ganciclovir results in death of the hypoimmunogenic cell.Hypoimmunogenic cell depletion may be accomplished by introducing a socall “suicide gene” via genome editing applications, e.g., ZFN,CRISPR/cas and TALEN systems.

Agents such as ganciclovir which mediate killing of a cell uponexpression of a gene such as thymidine kinase, are referred to herein as“cell death inducing agent.”

Genes which can be used to kill hypoimmunogenic cells include, but arenot limited to, herpes simplex virus thymidine kinase and cytosinedeaminase, or any gene which induces the death of a cell that can beplaced under the control of an inducible promoter/regulatory sequence(referred to interchangeably herein as a “promoter/regulatory sequence”or as a “promoter”). The gene is transferred into a hypoimmunogeniccell, the cells are selected under an appropriate selective pressure,the cells are transferred to the patient, and are allowed to engrafttherein. The patient is then treated with an agent, which inducespromoter activity, thereby inducing expression of the gene whose productfunctions to kill hypoimmunogenic cells. In the case of thymidinekinase, other agents which facilitate killing of the cell by this enzymemay also be used, such as, for example, ganciclovir (Bonini et al.,1997, Science 276:1719-1724; Bordignon et al., 1995, Human Gene Therapy6:813-819; Minasi et al., 1993, J. Exp. Med. 177:1451-1459; Braun etal., 1990, Biology of Reproduction 43:684-693). Other genes useful forthis purpose include, but are not limited to, constitutively activeforms of caspases 3, 8, and 9, bax, granzyme, diphtheria toxin,Pseudomonas A toxin, ricin and other toxin genes are disclosed elsewhereherein. The generation of appropriate constructs for delivery of suchgenes to a human will be readily apparent to the skilled artisan and isdescribed, for example, in Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York) and inAusubel et al. (1997, Current Protocols in Molecular Biology, John Wiley& Sons, New York).

It is important that the gene which is transferred into thehypoimmunogenic cells, for the purpose of killing the cells, be placedunder the control of the appropriate promoter sequence, such thatinduction of expression of the gene may be effected upon addition to thecells (administration to the mammal) of the appropriate inducer. Suchinducible promoter sequences include, but are not limited to promoterswhich are induced upon addition of a metal to the cells, steroidinducible promoters and the like. In one preferred embodiment, theecdysone promoter system may be employed. In this embodiment, theecdysone promoter is cloned upstream of the ecdysone receptor proteinsequence, which is positioned upstream of a second promoter sequencewhich drives expression of the ecdysone binding site operably linked tothe desired gene, for example, the desired toxin. Induction of thepromoter induces expression of the toxin, thereby effecting killing ofthe cell in which the toxin gene resides.

Cells which have transduced therein a gene for cell killing, when suchcells are transduced in an ex vivo manner, may be selected (i.e.,separated from cells which do not comprise the gene) by providing thecells with a selectable marker in addition to the transduced gene.Selectable markers are well known in the art and are described, forexample, in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y.).

Hypoimmunogenic cell depletion may further be accomplished byintroducing into a population of hypoimmunogenic cells anoligonucleotide (for example, but not limited to, an antisense molecule)or a ribozyme, which oligonucleotide or ribozyme is capable of inducingdeath of the hypoimmunogenic cell, or of inducing impairment ofhypoimmunogenic cell function. Such oligonucleotides include those whichtarget an essential function of an hypoimmunogenic cell, defined hereinas being one which either kills a hypoimmunogenic cell or impairs thefunction of the hypoimmunogenic cell with respect to stimulation of Tcells. Such functions of a hypoimmunogenic cell include, but are notlimited to, the costimulatory function of B71 and B72, CD40, amongothers. Thus, oligonucleotides and ribozymes which are useful in themethods of the invention include, but are not limited to, those whichare directed against these targets.

As noted herein, depletion of hypoimmunogenic cell includes impairmentof hypoimmunogenic cell function. Impairment of hypoimmunogenic cellfunction includes all forms of hypoimmunogenic cell impairment with orwithout physical removal or depletion of hypoimmunogenic cell. Thus,impairment of hypoimmunogenic cell function includes the use of anantibody that blocks the function of hypoimmunogenic cell surfacemolecules which are critical for hypoimmunogenic cell function.

Alternatively, peptides which block the function of hypoimmunogenic cellsurface molecules, which blocking results in impairment ofhypoimmunogenic cell function, may be used to effectively depletehypoimmunogenic cell in a host organism. Such peptides include, but arenot limited to, those which are designed to specifically bind receptormolecules on the surface of hypoimmunogenic cells, and those which aredesigned to, for example, inhibit essential enzymatic functions in thesecells.

Similarly, genes and oligonucleotides which are designed for the samepurpose as described herein, are also included as tools in the methodsof the invention. Thus, peptides, oligonucleotides and genes whichimpair the biological function of a hypoimmunogenic cell, as that termis defined herein, are also contemplated for use in the methods of theinvention disclosed herein.

The invention further encompasses the use of pharmaceutical compositionsof an appropriate hypoimmunogenic cell depleting composition to practicethe methods of the invention, the compositions comprising an appropriatehypoimmunogenic cell depleting composition and apharmaceutically-acceptable carrier. In some embodiments, the celldepleting composition is a chimeric composition comprising an antibodyand a toxin.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate hypoimmunogenic celldepleting composition may be combined and which, following thecombination, can be used to administer the appropriate hypoimmunogeniccell depleting composition to a mammal.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the hypoimmunogenic cell depleting composition, suchpharmaceutical compositions may contain pharmaceutically-acceptablecarriers and other ingredients known to enhance and facilitate drugadministration. Other possible formulations, such as nanoparticles,liposomes, resealed erythrocytes, and immunologically based systems mayalso be used to administer an appropriate hypoimmunogenic cell depletingcomposition according to the methods of the invention.

Methods of introducing “suicide genes” into cells are disclosed inUS20060222633 which is herein incorporated by reference in its entirety.

The invention includes a method of depleting hypoimmunogenic cells in amammalian host. After the hypoimmunogenic cells have been transplantedinto a host, the method comprises contacting the hypoimmunogenic cellswith a cell depleting composition to effect impairment ofhypoimmunogenic cell function or killing of the hypoimmunogenic cell,thereby depleting the hypoimmunogenic cells in the mammalian host.

In another aspect, the hypoimmunogenic cell depleting composition isselected from the group consisting of a toxin, an antibody, aradioactive molecule, a nucleic acid, a peptide, a peptidomemetic and aribozyme.

In one aspect, the toxin is an immunotoxin. The toxin is selected fromthe group consisting of ricin, diptheria toxin and pseudomonas exotoxinA.

In another embodiment, the antibody is selected from the groupconsisting of antibody specific for CD1a, antibody specific for CD11c,antibody specific for MHCII, antibody specific for CD11b, antibodyspecific for DEC205, antibody specific for B71, antibody specific forB72, antibody specific for CD40, antibody specific for Type I lectinsand antibody specific for Type II lectins.

In yet another embodiment, the nucleic acid molecule is selected fromthe group consisting of a gene and an oligonucleotide.

In a further embodiment, the radioactive molecule is a radioactivelylabeled antibody.

In another embodiment, the antigen depleting composition is a chimericcomposition comprising an antibody and a toxin. The toxin may beselected from the group consisting of ricin, diptheria toxin andpseudomonas exotoxin A.

In another embodiment, the antibody is selected from the groupconsisting of f antibody specific for CD1a, antibody specific for CD11c,antibody specific for MHCII, antibody specific for CD11b, antibodyspecific for DEC205, antibody specific for B71, antibody specific forB72, antibody specific for CD40, antibody specific for Type I lectinsand antibody specific for Type II lectins.

Combination Product

The embodiments described herein also disclose a combination product,which refers to a device loaded with hypoimmunogenic cells ortherapeutic agent, i.e. each alone may be a candidate medical device orcell product, but used together they make a combination product. In oneembodiment, the combination product refers to a perforated device loadedwith hypoimmunogenic cells. This is referred to as a “combinationproduct”, or “perforated combination product.” The device (perforated ornot) can be any macro cell delivery device described herein includingbut not limited to those cell encapsulation devices as described in U.S.Pat. Nos. 8,278,106 and 9,526,880, PCT Application No.PCT/US2016/0061442 and U.S. Design Patent Nos. D714956, D718472,D718467, D718466, D718468, D718469, D718470, D718471, D720469, D726306,D726307, D728095, D734166, D734847, D747467, D747468, D747798, D750769,D750770, D755986, D760399, D761423, D761424 (incorporated by referencein their entirety). The cells loaded into the device (perforated or not)may be any hypoimmunogenic cells discussed above including but notlimited to definitive endoderm, PDX1-positive endoderm, PDX1-positiveforegut endoderm, pancreatic endoderm, pancreatic endoderm cellsexpressing PDX1 and NKX6.1, endocrine progenitors, endocrine progenitorsexpressing NKX6.1 and INS, immature beta cell, immature beta cellsexpressing NKX6.1, INS and MAFB, mature endocrine cells, matureendocrine cells expressing INS, GCG, SST and PP, and mature beta cellsand mature beta cells expressing INS and MAFA.

Perforated delivery devices loaded with pancreatic endoderm orpancreatic progenitor hypoimmunogenic cells which mature when implantedin vivo are intended to reduce insulin dependence and/or reducehypoglycemia in patients with diabetes. This includes, but is notlimited to high-risk type I diabetic patients who are hypoglycemiaunaware, labile (brittle), or have received an organ transplant and whocan tolerate, or are already on, immune suppression therapy. Assubstantially described in PCT Application No. PCT/US2016/0061442(incorporated by reference in its entirety), the primary method ofaction is via human pancreatic endoderm cells (PEC) or pancreaticprogenitor hypoimmunogenic cells, contained in a permeable, durable,implantable medical device that facilitates direct host vascularization.The PEC hypoimmunogenic cells differentiate and mature into therapeuticglucose-responsive, insulin-releasing hypoimmunogenic cells afterimplantation. As such, the perforated combination product supportssecretion of human insulin. The perforated combination product limitsdistribution (egress) of PEC hypoimmunogenic cells in vivo. Theperforated combination product will be implanted in a location thatpermits sufficient vascular engraftment to sustain the population oftherapeutic hypoimmunogenic cells within the device and facilitatedistribution of insulin and other pancreatic products to thebloodstream. The perforated combination product is intended to beimplanted and explanted with conventional surgical tools, and to providea therapeutic dose for two years or more. The device is intended toretain an adequate dose of the PEC hypoimmunogenic cells product duringformulation, shelf-life, handling and surgical implant to achieveclinical efficacy and ensure the cell product is located within thetissue capsule to meet safety requirements.

Knock-in

In certain embodiments, tolerogenic factors can be inserted orreinserted into genome-edited stem cell lines to createimmune-privileged universal donor stem cell lines. In certainembodiments, the universal stem cells disclosed herein have been furthermodified to express one or more tolerogenic factors. Exemplarytolerogenic factors include, without limitation, one or more of HLA-C,HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, and IL-35.Any method of gene editing can be used to facilitate the insertion oftolerogenic factors, such as the tolerogenic factors above, into anAAVS1 locus, to actively inhibit immune rejection.

Specifically, in certain embodiments, the inventions disclosed hereinrelate to a stem cell, the genome of which has been altered to reduce ordelete critical components of at least one MHC-Class I gene and at leastone NK activating ligand gene and which has been further altered toincrease expression of one or more tolerogenic factors. In certainembodiments, the inventions disclosed herein relate to a stem cell, thegenome of which has been altered to reduce or delete critical componentsof at least one MHC-Class I gene and at least one NK activating ligandgene and which has been further altered to increase expression of one ormore of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47,CI-inhibitor, and IL-35.

Embodiments

Other embodiments of the invention are described with reference to thenumbered paragraphs below.

Related to Blocking Antibody: Composition

Paragraph 1: A composition comprising a pluripotent derived cell thatlacks at least one human leucocyte antigen (HLA)-Class I gene and atleast one agent that binds to a Natural Killer (NK) cell activatingligand.

Paragraph 2: The composition of paragraph 1, wherein the agent is anantibody.

Paragraph 3: The composition of paragraph 1, wherein the HLA-Class Igene is B2M.

Paragraph 4: The composition of paragraphs 1-3, wherein the NK cellactivating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or combinationsthereof.

Paragraph 5: The composition of paragraphs 1-2, wherein the NK cellactivating ligand is ICAM-1 and CEACAM1.

Paragraph 6: The composition of paragraphs 1-2, wherein the NK cellactivating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 7: The composition of paragraphs 1-2, wherein the pluripotentderived cells further express a protein which when expressed in thepresence of a cell death inducing agent, the agent is capable of killingthe pluripotent cells.

Paragraph 8: The composition of paragraph 7, wherein the cell deathinducing agent is ganciclovir.

Paragraph 9: The composition of any one of paragraphs 1-8, wherein thepluripotent derived cells further overexpress one or more tolerogenicfactors.

Paragraph 10: The composition of paragraph 9, wherein the tolerogenicfactors are HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor,or IL-35.

Paragraph 11: The composition of paragraph 9, wherein the tolerogenicfactors are HLA-C, HLA-E and HLA-G.

Related to Blocking Antibody: Method

Paragraph 1: A method of preventing cellular graft rejection of humanpluripotent derived cells, comprising administering to a subject in needof treatment a composition comprising a target cell population thatlacks at least one HLA-Class I gene, and at least one agent that bindsan NK cell activating ligand on the target cell in an amount effectiveto suppress the subject's NK cell attack thereby preventing cellulargraft rejection of human pluripotent derived cells.

Paragraph 2: The method of paragraph 1, wherein the agent is anantibody.

Paragraph 3: The method of paragraph 1, wherein the subject's immuneresponse to a NK cell activating ligand is suppressed.

Paragraph 4: The method of paragraph 1, wherein the NK cell activatingligand is ICAM-1, CEACAM1, CADM1, MICA or MICB.

Paragraph 5: The method of paragraph 1, wherein the NK cell activatingligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 6: The method of paragraph 1, wherein the subject is human.

Paragraph 7: The method of paragraph 2, wherein the antibody is a humanantibody.

Related to hES Cell Double Knockout: Compositions

Paragraph 1: An in vitro cell population comprising pluripotent derivedcells, wherein the pluripotent derived cells lack at least one HLA-ClassI gene and at least one Natural killer (NK) cell activating ligand gene.

Paragraph 2: The in vitro cell population of paragraph 1, wherein theHLA-Class I gene is B2M.

Paragraph 3: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB orcombinations thereof.

Paragraph 4: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1 and CEACAM1.

Paragraph 5: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 6: The in vitro cell population of paragraph 1-5, wherein thepluripotent derived cells further express a protein which when expressedin the presence of a cell death inducing agent, the agent is capable ofkilling the pluripotent derived cells.

Paragraph 7: The in vitro cell population of paragraph 6, wherein thecell death inducing agent is ganciclovir.

Paragraph 8: The in vitro cell population of any one of paragraphs 1-7,wherein the derived pluripotent cells further overexpress one or moretolerogenic factors.

Paragraph 9: The in vitro cell population of paragraphs 8, wherein thetolerogenic factors are HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47,CI-inhibitor, IL-35 or combinations thereof.

Paragraph 10: The in vitro cell population of paragraphs 9, wherein thetolerogenic factors are HLA-C, HLA-E and HLA-G.

Related to Pluripotent Stem Cells Triple Knockout: Compositions

A human pluripotent stem cell comprising a modified genome comprising: afirst genomic modification in which the B2M gene has been edited toreduce or eliminate B2M surface expression and/or activity in the cell;(b) a second genomic modification in which the ICAM-1 gene has beenedited to reduce or eliminate ICAM-1 surface expression and/or activityin the cell; and (c) a third genomic modification in which the CEACAM1gene has been edited to reduce or eliminate CEACAM1 surface expressionand/or activity in the cell.

Related to Knockout of Transcriptional Regulators: Compositions

Paragraph 12: A pluripotent derived cell comprising modulated expressionof one or more MHC-Class I or MHC-Class II genes or protein complexesand one or more NK cell activating ligands relative to a wild-typepluripotent stem cell, wherein the pluripotent stem cell has one or moregenes encoding one or more transcriptional regulators of MHC-Class I orMHC-Class II and NK cell activating ligand genes deleted from at leastone allele of the cell.

Paragraph 13: A pluripotent stem cell comprising modulated expression ofone or more NK cell activating ligands relative to a wild-type humanpluripotent stem cell.

Paragraph 14: A human pluripotent stem cell that does not express B2M orICAM-1.

Paragraph 15: A human pluripotent stem cell that does not express CIITAor ICAM-1.

Paragraph 16: A human pluripotent stem cell that does not express LRCSor ICAM-1.

Paragraph 17: A human pluripotent stem cell that does not express one ormore of NLRCS, CIITA and B2M and further does not express one or more ofICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 18: A human pluripotent stem cell that does not express one ormore of HLA-A, HLA-B and HLA-C and further does not express one or moreof ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 19: A human pluripotent stem cell that does not express one ormore of one or more MHC-Class I antigens and one or more NK cellactivating ligands and further has one or more tolerogenic factorsinserted into a safe harbor locus of at least one allele of the cell.

Paragraph 20: A human pluripotent stem cell comprising a modified genomecomprising a first genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell and in which the ICAM-1 gene has been edited to reduce oreliminate ICAM-1 surface expression and/or activity in the cell.

Paragraph 21: A human pluripotent stem cell comprising a modified genomecomprising: a first genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell; and (b) a second genomic modification in which the ICAM-1 genehas been edited to reduce or eliminate ICAM-1 surface expression and/oractivity in the cell.

Related to hES double knockout cells: compositions Paragraph 1: An invitro cell population comprising pluripotent cells, wherein thepluripotent cells lack at least one functional MHC-Class I cell surfaceprotein and at least one functional Natural killer (NK) cell activatingligand cell surface protein.

Paragraph 2: The in vitro cell population of paragraph 1, wherein theMHC-Class I cell surface protein is HLA-A, HLA-B, HLA-C or combinationsthereof.

Paragraph 3: The in vitro cell population of paragraphs 1-2, wherein theMHC-Class I cell surface protein is B2M.

Paragraph 4: The in vitro cell population of any one of paragraphs 1-3,wherein the pluripotent cells lack at least two functional NK cellactivating ligand cell surface proteins.

Paragraph 5: The in vitro cell population of any one of paragraphs 1-4,wherein the pluripotent cells lack at least three functional NK cellactivating ligand cell surface proteins.

Paragraph 6: The in vitro cell population of any one of paragraphs 1-5,wherein NK cell activating ligand cell surface protein is ICAM-1,CEACAM1, CADM1, MICA, MICB or combinations thereof.

Paragraph 7: The in vitro cell population of any one of paragraphs 1-5,wherein the NK cell activating ligand cell surface protein is ICAM-1 andCEACAM1.

Paragraph 8: The in vitro cell population of any one of paragraphs 1-7,wherein the pluripotent cells further express a protein which whenexpressed in the presence of a cell death inducing agent, the agent iscapable of killing the pluripotent cells.

Paragraph 9: The in vitro cell population of any one of paragraphs 1-8,wherein the protein which when expressed in the presence of a cell deathinducing agent, the agent is capable of killing the pluripotent cells isherpes simplex virus, thymidine kinase or cytosine deaminase.

Paragraph 10: The in vitro cell population of any one of paragraphs 8-9,wherein the cell death inducing agent is ganciclovir.

Related to hES Double Knockout Cells: Compositions

Paragraph 1: An in vitro cell population comprising pluripotent cells,wherein the pluripotent cells have reduced expression of at least oneMHC-Class I cell surface protein and reduced function and/or expressionof at least one NK activating ligand cell surface protein relative tothe original genotype or relative to a wild-type human cell.

Paragraph 2: An in vitro cell population comprising pluripotent cells,wherein the pluripotent cells have reduced expression of one or more ofHLA-A, HLA-B and HLA-C cell surface protein and reduced function and/orexpression of at least one NK activating ligand cell surface proteinrelative to the original genotype or relative to a wild-type human cell.

Paragraph 3: An in vitro cell population comprising pluripotent cells,wherein the pluripotent cells lack functional HLA cell surface proteinexpression, NK activating ligand cell surface protein expression andhave a protein which when expressed in the pluripotent cells in thepresence of the cell death inducing agent, the agent is capable ofkilling the pluripotent cells.

Paragraph 4: A stem cell wherein expression of one or more HLA-Class Icell surface protein and one or more NK activating ligand cell surfaceprotein is modulated relative to a wild-type stem cell.

Paragraph 5: A pluripotent cell wherein expression of one or moreHLA-Class I cell surface protein, one or more NK activating ligand cellsurface protein, and one or more tolerogenic cell surface proteinfactors is modulated relative to a wild-type pluripotent cell andwherein the pluripotent cells further express a protein which whenexpressed in the presence of a cell death inducing agent, the agent iscapable of killing the pluripotent cells.

Paragraph 6: An in vitro cell population comprising pluripotent cells,wherein the pluripotent cells lack functional MHC-Class I genes andNatural killer (NK) cell activating ligand genes and wherein thepluripotent cells overexpress tolerogenic cell surface protein factorsrelative to a wild-type pluripotent cell and wherein the pluripotentcells further express a protein which when expressed in the presence ofa cell death inducing agent, the agent is capable of killing thepluripotent cells.

Related to PEC Double Knockout Cells: Composition

Paragraph 1: An in vitro cell population comprising pancreatic endoderm(PEC) cells, wherein the PEC cells lack at least one functionalHLA-Class I gene and at least one Natural killer (NK) cell activatingligand gene.

Paragraph 2: The in vitro cell population of paragraph 1, wherein the atleast one HLA-Class I gene is B2M.

Paragraph 3: The in vitro cell population of paragraph 1-2, wherein theat least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA,MICB or combinations thereof.

Paragraph 4: The in vitro cell population of paragraph 1-2, wherein theat least one NK cell activating ligand is ICAM-1 and CEACAM1.

Paragraph 5: The in vitro cell population of paragraph 1-2, wherein theat least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICAand MICB.

Paragraph 6: The in vitro cell population of paragraph 1-5, wherein thePEC cells further express a protein which when expressed in the presenceof a cell death inducing agent, the agent is capable of killing the PECcells.

Paragraph 7: The in vitro cell population of paragraph 6, wherein thecell death inducing agent is ganciclovir.

Paragraph 8: The in vitro cell population of paragraph 6, wherein thegene which when expressed in the presence of a cell death inducingagent, the agent is capable of killing the PEC cells, is herpes simplexvirus, thymidine kinase or cytosine deaminase.

Paragraph 9: The in vitro cell population of any one of paragraphs 1-8,wherein the PEC cells further overexpress one or more tolerogenic cellsurface proteins.

Paragraph 10: The in vitro cell population of paragraphs 1-9, whereinthe tolerogenic factors are HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47,CI-inhibitor, IL-35 or combinations thereof.

Paragraph 11: The in vitro cell population of paragraphs 1-9, whereinthe tolerogenic factors are HLA-C, HLA-E and HLA-G.

Paragraph 12: An in vitro cell population comprising pancreatic endoderm(PEC) cells, wherein the PEC cells lack at least one functionalMHC-Class I gene, MHC-Class II gene and Natural killer (NK) cellactivating ligand gene.

Paragraph 13: An in vitro cell population comprising pancreatic endoderm(PEC) cells, wherein the PEC cells lack at least one functionalMHC-Class I gene and MHC-Class II gene and lack at least two Naturalkiller (NK) cell activating ligand genes.

Paragraph 14: A pancreatic endoderm (PEC) cell wherein one or moreHLA-Class I gene and one or more NK activating ligand gene is modulatedrelative to a wild-type PEC cell.

Paragraph 15: A pancreatic endoderm (PEC) cell wherein expression of oneor more HLA-Class I cell surface proteins, one or more NK activatingligand, and one or more tolerogenic factors is modulated relative to awild-type PEC cell and wherein the pancreatic endoderm cell furtherexpress a protein which when expressed in the presence of a cell deathinducing agent, the agent is capable of killing the PEC cells.

Paragraph 16: A pancreatic endoderm (PEC) cell that does not express B2Mor ICAM-1.

Paragraph 17: A pancreatic endoderm (PEC) cell that does not expressCIITA or ICAM-1.

Paragraph 18: A pancreatic endoderm (PEC) cell that does not expressLRCS or ICAM-1.

Paragraph 19: A pancreatic endoderm (PEC) cell that does not express oneor more of NLRCS, CIITA and B2M and further does not express one or moreof ICAM-1, CEACAM1, CADM1, MICa and MICB.

Paragraph 20: A pancreatic endoderm (PEC) cell that does not express oneor more of HLA-A, HLA-B and HLA-C and further does not express one ormore of ICAM-1, CEACAM1, CADM1, MICa and MICB.

Paragraph 21: A pancreatic endoderm (PEC) cell that does not express oneor more MHC-Class I cell surface proteins and one or more NK cellactivating ligands and further has one or more tolerogenic factorsinserted into a safe harbor locus of at least one allele of the cell.

Paragraph 22: A pancreatic endoderm (PEC) cell comprising a modifiedgenome comprising a first genomic modification in which the B2M gene hasbeen edited to reduce or eliminate B2M surface expression and/oractivity in the cell and in which the ICAM-1 gene has been edited toreduce or eliminate ICAM-1 surface expression and/or activity in thecell.

Paragraph 23: A pancreatic endoderm (PEC) cell comprising a modifiedgenome comprising: a first genomic modification in which the B2M genehas been edited to reduce or eliminate B2M surface expression and/oractivity in the cell; and (b) a second genomic modification in which theICAM-1 gene has been edited to reduce or eliminate ICAM-1 surfaceexpression and/or activity in the cell.

Paragraph 24: A pancreatic endoderm (PEC) cell comprising a modifiedgenome comprising: a first genomic modification in which the B2M genehas been edited to reduce or eliminate B2M surface expression and/oractivity in the cell; (b) a second genomic modification in which theICAM-1 gene has been edited to reduce or eliminate ICAM-1 surfaceexpression and/or activity in the cell; and (c) a third genomicmodification in which the CEACAM1 gene has been edited to reduce oreliminate CEACAM1 surface expression and/or activity in the cell.

Related to Hypoimmunogenic Cells

Paragraph 1: An in vitro cell population comprising hypoimmunogeniccells, wherein the hypoimmunogenic cells lack at least one functionalHLA-Class I cell surface protein and at least one functional NK cellactivating ligand cell surface protein.

Paragraph 2: The in vitro cell population of paragraph 1, wherein theHLA-Class I cell surface protein is B2M.

Paragraph 3: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB orcombinations thereof.

Paragraph 4: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1 and CEACAM1.

Paragraph 5: The in vitro cell population of paragraph 1-2, wherein theNK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 6: The in vitro cell population of paragraph 1-5, wherein thehypoimmunogenic cells further express a protein which when expressed inthe presence of a cell death inducing agent, the agent is capable ofkilling the hypoimmunogenic cells.

Paragraph 7: The in vitro cell population of any one of paragraphs 1-6,wherein the hypoimmunogenic cells further overexpresses one or moretolerogenic factors.

Paragraph 8: The in vitro cell population of paragraphs 1-7, wherein thetolerogenic factors are HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47,CI-inhibitor, IL-35 and combinations thereof.

Paragraph 9: The in vitro cell population of paragraphs 1-7, wherein thetolerogenic factors are HLA-C, HLA-E and HLA-G.

Paragraph 10: An in vitro cell population comprising hypoimmunogeniccells, wherein the hypoimmunogenic cells lack at least one functionalMHC-Class I gene and at least one NK cell activating ligand gene.

Paragraph 11: An in vitro cell population comprising hypoimmunogeniccells, wherein the hypoimmunogenic cells lack at least one functionalMHC-Class I gene, MHC-Class II gene and NK cell activating ligand gene.

Paragraph 12: The in vitro cell population of paragraphs 10 or 11,wherein the MHC-Class I gene is HLA-A, HLA-B, HLA-C or combinationsthereof.

Paragraph 13: The in vitro cell population of paragraphs 10, 11 or 12,wherein the MHC-Class I gene is B2M.

Paragraph 14: The in vitro cell population of any one of paragraphs10-13, wherein the hypoimmunogenic cells lack at least two functional NKcell activating ligand genes.

Paragraph 15: The in vitro cell population of any one of paragraphs10-13, wherein the hypoimmunogenic cells lack at least three functionalNK cell activating ligand genes.

Paragraph 16: The in vitro cell population of any one of paragraphs10-13, wherein NK cell activating ligand is ICAM-1, CEACAM1, CADM1,MICA, MICB or combinations thereof.

Paragraph 17: The in vitro cell population of any one of paragraphs10-13, wherein the NK cell activating ligand is ICAM-1 and CEACAM1.

Paragraph 18: The in vitro cell population of any one of paragraphs10-13, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1,MICA and MICB.

Paragraph 19: The in vitro cell population of any one of paragraphs10-18, wherein the hypoimmunogenic cells further express a protein whichwhen expressed in the presence of a cell death inducing agent, the agentis capable of killing the hypoimmunogenic cells.

Paragraph 20: The in vitro cell population of any one of paragraphs7-15, wherein the hypoimmunogenic cells are hES cells or pancreaticlineage cells.

Paragraph 21: A hypoimmunogenic cell wherein expression of one or moreHLA-Class I cell surface protein and one or more NK activating ligandcell surface protein is modulated relative to a wild-typehypoimmunogenic cells.

Paragraph 22: A hypoimmunogenic cell wherein expression of one or moreHLA-Class I cell surface protein, one or more NK activating ligand, andone or more tolerogenic cell surface protein factors is modulatedrelative to a wild-type hypoimmunogenic cell and wherein the pluripotentcells further express a protein which when expressed in the presence ofa cell death inducing agent, the agent is capable of killing thehypoimmunogenic cells.

Paragraph 23: A hypoimmunogenic stem cell comprising modulatedexpression of one or more MHC-Class I or MHC-Class II cell surfaceproteins and one or more NK cell activating ligands relative to awild-type pluripotent stem cell, wherein the pluripotent stem cell hasone or more genes encoding one or more transcriptional regulators ofMHC-Class I or MHC-Class II and NK cell activating ligand genes deletedfrom at least one allele of the cell.

Paragraph 24: A hypoimmunogenic cell comprising modulated expression ofone or more NK cell activating ligands relative to a wild-type humanhypoimmunogenic cell.

Paragraph 25: A human hypoimmunogenic cell that does not express B2M orICAM-1.

Paragraph 26: A human hypoimmunogenic cell that does not express CIITAor ICAM-1.

Paragraph 27: A human hypoimmunogenic cell that does not express LRCS orICAM-1.

Paragraph 28: A human hypoimmunogenic cell that does not express one ormore of NLRCS, CIITA and B2M and further does not express one or more ofICAM-1, CEACAM1, CADM1, MICa and MICB.

Paragraph 29: A human hypoimmunogenic cell that does not express one ormore of HLA-A, HLA-B and HLA-C and further does not express one or moreof ICAM-1, CEACAM1, CADM1, MICa and MICB.

Paragraph 30: A human hypoimmunogenic cell that does not express one ormore of one or more MHC-Class I cell surface proteins and one or more NKcell activating ligands and further has one or more tolerogenic factorsinserted into a safe harbor locus of at least one allele of the cell.

Paragraph 31: A hypoimmunogenic cell comprising a modified genomecomprising a first genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell and in which the ICAM-1 gene has been edited to reduce oreliminate ICAM-1 surface expression and/or activity in the cell.

Paragraph 32: A hypoimmunogenic stem cell comprising a modified genomecomprising: a first genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell; and (b) a second genomic modification in which the ICAM-1 genehas been edited to reduce or eliminate ICAM-1 surface expression and/oractivity in the cell.

Paragraph 33: A hypoimmunogenic stem cell comprising a modified genomecomprising: a first genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell; (b) a second genomic modification in which the ICAM-1 gene hasbeen edited to reduce or eliminate ICAM-1 surface expression and/oractivity in the cell; and (c) a third genomic modification in which theCEACAM1 gene has been edited to reduce or eliminate CEACAM1 surfaceexpression and/or activity in the cell.

Methods for Double Knockout

Paragraph 1: A method of reducing graft rejection, comprising:

a) administering to a subject in need of a transplant, an effectiveamount of a graft comprising a pancreatic endoderm cell populationwherein the function of at least one HLA-Class I cell surface proteinand at least one NK cell activating ligand cell surface protein isdisrupted.

Paragraph 2: The method of paragraph 1, wherein the HLA-Class I cellsurface protein is B2M.

Paragraph 3: The method of paragraph 1-2, wherein the NK cell activatingligand cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB orcombinations thereof.

Paragraph 4: The method of paragraph 1-2, wherein the NK cell activatingligand is ICAM-1 and CEACAM1.

Paragraph 5: The method of paragraph 1-2, wherein the NK cell activatingligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

Paragraph 6: A method of depleting hypoimmunogenic cells in a populationof cells, said method comprising contacting said hypoimmunogenic cellswith a hypoimmunogenic cell depleting composition to effect impairmentof the hypoimmunogenic cell function or killing of said hypoimmunogeniccells, thereby depleting said hypoimmunogenic cells in said populationof cells.

Paragraph 7: The method of claim 6, wherein the hypoimmunogenic cellslack at least one functional HLA-Class I cell surface protein and atleast one NK activating ligand expression.

Paragraph 8: A method of removing hypoimmunogenic cells from a hostmammal, said method comprising: (a) transferring hypoimmunogenic cellsto said host mammal; and (b) contacting the host with a hypoimmunogeniccell depleting composition to effect impairment of the hypoimmunogeniccell function or killing of said hypoimmunogenic cells, thereby removingsaid hypoimmunogenic cells in the host mammal.

Paragraph 9: The method of paragraph 8, wherein the function of at leastone HLA-Class I cell surface protein and at least one NK activatingligand is diminished.

Paragraph 10: A method of increasing NK activating ligands in a targetcell population comprising exposing the target cell population to IFN-γstimulation thereby increasing NK activating ligands in a target cellcompared to wild type.

A method of preparing a hypoimmunogenic stem cell, the method comprisingmodulating expression of one or more MHC-Class I cell surface proteinsand one or more NK activating ligands by the hypoimmunogenic stem celland thereby preparing the hypoimmunogenic stem cell.

A method of preparing a hypoimmunogenic stem cell, the method comprisingmodulating expression of one or more MHC-Class I cell surface protein,one or more NK activating ligand and modulating expression of one ormore tolerogenic factors on the stem cell and thereby preparing thehypoimmunogenic stem cell.

A method of modulating expression of one or more MHC-Class I cellsurface proteins and NK cell activating ligand cell surface proteins ona stem cell, comprising deleting one or more genes encoding one or moretranscriptional regulators of MHC-Class I genes and NK cell activatingligands from at least one allele of the cell and thereby modulatingexpression of the one or more MHC-Class I cell surface proteins and NKcell activating ligand cell surface proteins.

A hypoimmunogenic stem cell comprising a modified genome comprising (a)a first genomic modification in which the B2M gene has been edited toreduce or eliminate B2M surface expression and/or activity in the cellby contacting the cell with a Cas protein or a nucleic acid encoding aCas protein and a ribonucleic acid comprising a sequence of any one ofSEQ ID NOs: 1-3; and (b) a second genomic modification in which theICAM-1 gene has been edited to reduce or eliminate ICAM-1 surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding a Cas protein and a ribonucleic acidcomprising a sequence of any one of SEQ ID NOs: 4-6.

A hypoimmunogenic stem cell comprising a modified genome comprising (a)a first genomic modification in which the B2M gene has been edited toreduce or eliminate B2M surface expression and/or activity in the cellby contacting the cell with a Cas protein or a nucleic acid encoding aCas protein and a ribonucleic acid comprising a sequence of any one ofSEQ ID NOs: 1-3; (b) a second genomic modification in which the ICAM-1gene has been edited to reduce or eliminate ICAM-1 surface expressionand/or activity in the cell by contacting the cell with a Cas protein ora nucleic acid encoding a Cas protein and a ribonucleic acid comprisinga sequence of any one of SEQ ID NOs: 4-6; and (c) a third genomicmodification in which the CEACAM1 gene has been edited to reduce oreliminate CEACAM1 surface expression and/or activity in the cell bycontacting the cell with a Cas protein or a nucleic acid encoding a Casprotein and a ribonucleic acid comprising a sequence of any one of SEQID NOs: 7-9.

A pluripotent stem cell comprising a modified genome comprising (a) afirst genomic modification in which the B2M gene has been edited toreduce or eliminate B2M surface expression and/or activity in the cellby contacting the cell with a Cas protein or a nucleic acid encoding aCas protein and a ribonucleic acid comprising a sequence of any one ofSEQ ID NOs: 1-3; and (b) a second genomic modification in which theICAM-1 gene has been edited to reduce or eliminate ICAM-1 surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding a Cas protein and a ribonucleic acidcomprising a sequence of any one of SEQ ID NOs: 4-6.

The sequences of SEQ ID Nos: 1-9 are provided below in Table 4, anddescribed below.

SEQ ID NO. 1: Exon 1, negative strand.

SEQ ID NO. 2: Exon 2, negative strand.

SEQ ID NO. 3: Exon 1, negative strand.

SEQ ID NO. 4: Exon 2, positive strand.

SEQ ID NO. 5: Exon 2, negative strand.

SEQ ID NO. 6: Exon 1, positive strand.

SEQ ID NO. 7: Exon 1, negative strand.

SEQ ID NO. 8: Exon 1, positive strand.

SEQ ID NO. 9: Exon 1, positive strand.

SEQ. ID Gene Sequences NO. B2M CGCGAGCACAGCTAAGGCCACGG 1 (beta-2-CAGTAAGTCAACTTCAATGTCGG 2 microglobulin) GAGTAGCGCGAGCACAGCTAAGG 3 ICAM1CCTCAAAAGTCATCCTGCCCCGG 4 (intercellular AGCAACTCCTTTTTAGGCAACGG 5adhesion CCGCACTCCTGGTCCTGCTCGGG 6 molecule 1) CEACAM1GAGTGCGTGTACCCTGGCAGGGG 7 (carcinoembryonic GGTACACGCACTCTGTGAAGTGG 8antigen related TACACGCACTCTGTGAAGTGGGG 9 cell adhesion molecule 1)

A pancreatic endoderm (PEC) cell comprising a modified genome comprising(a) a first genomic modification in which the B2M gene has been editedto reduce or eliminate B2M surface expression and/or activity in thecell by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a ribonucleic acid comprising a sequence ofany one of SEQ ID NOs: 1-3; (b) a second genomic modification in whichthe ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding a Cas protein and a ribonucleic acidcomprising a sequence of any one of SEQ ID NOs: 4-6; and (c) a thirdgenomic modification in which the CEACAM1 gene has been edited to reduceor eliminate CEACAM1 surface expression and/or activity in the cell bycontacting the cell with a Cas protein or a nucleic acid encoding a Casprotein and a ribonucleic acid comprising a sequence of any one of SEQID NOs: 7-9.

A method of reducing hypoglycemia, comprising:

a) administering to a subject in need of a transplant, an effectiveamount of a graft comprising a pancreatic endoderm cell populationwherein the function of at least one HLA-Class I cell surface proteinand at least one NK cell activating ligand cell surface protein isdisrupted wherein the pancreatic endoderm cell population matures invivo and produces insulin in response to glucose stimulation in vivo,thereby reducing hypoglycemia in a patient.

A method of reducing insulin dependence, comprising:

a) administering to a subject in need of a transplant, an effectiveamount of a graft comprising a pancreatic endoderm cell populationwherein the function of at least one HLA-Class I cell surface proteinand at least one NK cell activating ligand cell surface protein isdisrupted wherein the pancreatic endoderm cell population matures invivo and produces insulin in response to glucose stimulation in vivo,thereby reducing insulin dependence in a patient.

Definitions

“Hypoimmunogenic” or “universal donor cells” or “mutant cell” orequivalents thereof means a cell with reduced or eliminated expressionof at least one HLA-Class I cell surface protein and at least one NKactivating ligand. Such a cell is expected to be less prone to immunerejection or graft rejection by a subject into which such cells or graftare transplanted. For example, relative to an unaltered wild-type cell,such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immunerejection by a subject into which such cells are transplanted.

The term “treating” or “healing” or equivalents thereof refers to atherapeutic intervention that reduces (ameliorates) a sign or symptom.

The term “patient” or “host” or “mammalian host” or “subject” orequivalents thereof refers to living multi-cellular vertebrateorganisms, a category that includes both human and non-human mammals. Insome embodiments, the subject is a human subject. The preferred patientfor treatment is a human. The target patient populations may change overtime of clinical use/experience in ways that are independent of thecombination product itself, but rather related to the nature of theimmunosuppression regimen or lack thereof. For example, the combinationproduct might be used in a T1D population using a hypoimmunogenic celltherapy in combination with an immuno-suppressive drug (ISD) regimenthat achieves operational tolerance or is low in toxicity and sideeffect profile.

The term “blocking agent” herein refers to any agent capable of bindingto an NK activating ligand on the surface of a target cell including butnot limited to an antibody; or refers to an agent that prevents orinhibits protein expression of an NK activating ligand including but notlimited to a protein, an enzyme or chemical presently known or later tobe developed.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e. g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological blocking activity.

The term “antibody fragments” used herein refers to a portion of anintact antibody, preferably comprising the antigen-binding or variableregion thereof. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chainantibody molecules, and multispecific antibodies formed from antibodyfragments.

The term “blocking antibody” used herein refers to an antibody that,when it binds to an NK cell activating ligand on the target cell in vivoor in vitro, results in preventing or lessening the ability of the NKcell to lyse the target cell.

As used herein, the term “syngenic” or “syngeneic” refers to cells,tissues or organs that are genetically identical or are derived from agenetically identical source to the transplant recipient {e.g., anidentical twin), especially with respect to antigens or immunologicalreactions. Such cells, tissues or organs are called isografts. As usedherein, the term “allogenic” or “allogeneic” refers to cells, tissues ororgans that are not genetically identical or are derived from anon-genetically identical source to the transplant recipient (e.g., anon-related donor), especially with respect to antigens or immunologicalreactions. Such cells, tissues or organs are called allografts,allogeneic transplants, homografts or allotransplants.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “effective amount” or “therapeutically effective amount” orequivalents thereof refers to a quantity of an agent sufficient toachieve a desired effect in a subject or a cell being treated. Forinstance, this can be the amount of cells necessary to inhibit or tomeasurably reduce blood glucose levels and ultimately achievehomeostatic glycemic control. It can also mean an effective amount of anagent to change the function or structure of a cell or subject. Atherapeutically effective amount of an agent may be administered in asingle dose, or in several doses. However, the effective amount will bedependent on the particular agent applied, the subject being treated,the severity and type of the affliction, and the manner ofadministration.

The terms “decrease,” “disrupted,” “reduced,” “reduction,” and “inhibit”are all used herein generally to mean a decrease, specifically, decreaseby a statistically significant amount. However, for avoidance of doubt,“decreased,” “reduced,” “reduction,” “inhibited” includes a decrease byat least 10% as compared to a reference level, for example a decrease byat least about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90% or up to and including a 100%decrease (i.e. absent level as compared to a reference sample), or anydecrease between 10-100% as compared to a reference level, or at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold, or at least about a 10-fold decrease, or anydecrease between 2-fold and 10-fold or greater as compared to areference level.

The terms “increased,” “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, or up to and including a 100%increase, or any increase between 10-100% as compared to a referencelevel, or at least about a 2-fold, or at least about a 3-fold, or atleast about a 4-fold, or at least about a 5-fold, or at least about a10-fold increase, or any increase between 2-fold and 10-fold or greateras compared to a reference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of a reference. The term canalso mean two standard deviation (2SD) above normal, or higher,concentration of the reference. The term refers to statistical evidencethat there is a difference. It is defined as the probability of making adecision to reject the null hypothesis when the null hypothesis isactually true. The decision is often made using the p-value.

As used herein, “reduced hypoglycemia” or equivalents thereof means areduction in the number of hypoglycemic episodes together with nodeterioration in glycemic control, defined by <0.2% increase in HbAlc.

As used herein “reduced insulin dependence” or equivalents thereof meansa reduction in the number and/or dose of exogenous insulin injectionstogether with no deterioration in glycemic control, defined by <0.2%increase in HbAlc.

As used herein “tissue capsule” or equivalents thereof means the foreignbody capsule that forms around an implant or graft. The combinationproduct and/or device or perforated device containing the cells areintended to be retained within the capsule during the implant period.

“Engraftment” or equivalents thereof refers to differentiation of aprogenitor or immature cell population into a mature cell type. Forexample, engraftment of a PDX1-positive pancreatic endoderm cellpopulation maturing into a pancreatic endocrine cell population.

“Graft” refers to a differentiated cell population encapsulated ordelivered in the devices herein. For example, cell populations includingbut not limited to a pancreatic endoderm, a pancreatic progenitor, aPDX-1 positive pancreatic endoderm, a pancreatic endocrine precursor,pancreatic endocrine, singly or polyhormonal endocrine, pre-beta, beta,and/or insulin secreting grafts.

The term “essentially” or “substantially” or equivalents thereof meansmostly or a de minimus or a reduced amount of a component or cellpresent in any cell population or culture, e.g., immature beta cellcultures are “essentially or substantially immature beta cellsexpressing INS, NKX6.1 and PDX1 and not essentially or substantiallyexpressing NGN3”. Other examples include but not limited to “essentiallyor substantially hES cells”, “essentially or substantially definitiveendoderm cells”, “essentially or substantially foregut endoderm cells”,“essentially or substantially PDX1-negative foregut endoderm cells”,“essentially or substantially PDX1-positive pancreatic endoderm cells”,“essentially or substantially pancreatic endocrine precursor cells”,“essentially or substantially pancreatic endocrine cells” and the like.

With respect to cells in cell cultures or in cell populations, the term“substantially free of” or equivalents thereof means that the specifiedcell type of which the cell culture or cell population is free, ispresent in an amount of less than about 10%, less than about 9%, lessthan about 8%, less than about 7%, less than about 6%, less than about5%, less than about 4%, less than about 3%, less than about 2% or lessthan about 1% of the total number of cells present in the cell cultureor cell population.

The term “non-woven fabric” or equivalents thereof, includes, but is notlimited to, bonded fabrics, formed fabrics, or engineered fabrics, thatare manufactured by processes other than, weaving or knitting.

It is to be understood that the inventions disclosed herein are notlimited in their application to the details set forth in the descriptionor as exemplified. The invention encompasses other embodiments and iscapable of being practiced or carried out in various ways. Also, it isto be understood that the phraseology and terminology employed herein isfor the purpose of description and should not be regarded as limiting.

While certain compositions, methods and assays of the present inventionhave been described with specificity in accordance with certainembodiments, the following examples serve only to illustrate the methodsand compositions of the invention and are not intended to limit thesame.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims is introduced into another claim dependent onthe same base claim (or, as relevant, any other claim) unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. Where elementsare presented as lists, (e.g., in Markush group or similar format) it isto be understood that each subgroup of the elements is also disclosed,and any element(s) can be removed from the group. It should beunderstood that, in general, where the invention, or aspects of theinvention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not in every casebeen specifically set forth in so many words herein. It should also beunderstood that any embodiment or aspect of the invention can beexplicitly excluded from the claims, regardless of whether the specificexclusion is recited in the specification. The publications and otherreference materials referenced herein to describe the background of theinvention and to provide additional detail regarding its practice arehereby incorporated by reference.

EXAMPLES Example 1: Generation of B2M Deficient hES Cells

B2M deficient hES cells were generated using CyT49 cell line, however,any human pluripotent stem cell line can be used. Targeted disruption ofB2M gene generated cells, which do not express any HLA-Class I proteinson their cell surface. Both alleles of the B2M locus in the CyT49 hESCline were disrupted using CRISPR/Cas9 technology using known techniquesas outlined in PCT Publication No. WO2016183041A (which is incorporatedherein in its entirety). However, other nucleases including zinc-fingernucleases (ZFN) and transcription activator-like effector nuclease(TALEN) can be used to edit genes, as well traditional homologousrecombination and the like. Examples of published sequences for B2M aresubmitted as SEQ ID NOS.: 1, 2, and 3. NEXTGEN™ CRISPR (TransposagenInc., Lexington Ky.) was used to edit the gene, which incorporates dualguide RNA's and a catalytically inactive Cas9 protein fused to the FokInuclease. Plasmids containing the guide RNA's and Cas9 wereelectroporated into CyT49 hESC, and cells were seeded onto tissueculture plates. Twelve days post-electroporation, cells were sorted fornegative reactivity to a B2M antibody (BioLegend Cat#316306) byfluorescence activated cell sorting (FACS). Sorted cells were plated atclonal density. Individual clones were picked and plated at about day25. Clones were expanded and cryopreserved. Expanded clones that showeda normal karyotype by G-banding, and were negative for expression of B2Mprotein and for surface expression of HLA-class I proteins by flowcytometry and/or immunofluorescence were chosen for furtherexperimentation.

B2M surface expression in wild type (WT) and knockout cells was assessedby flow cytometry under normal and inflammatory conditions (afterexposure to interferon (IFN)-γ). See FIG. 2A: Normal: untreated growthmedia (Line B); inflammatory: exposed to 100 ng/mL of IFN-γ for 18-24hours (Line A) Inflammatory response occurs in relation to tissuetrauma. This releases proinflammatory cytokines, some of which areIL-1-α, TNF-β, IL-6, IL-8 and IFN-γ. Although WT and B2M−/− ESC and PEChave been treated with IFN-γ, these observations can be extended withother cytokines as well.

FIG. 2A shows B2M expression in WT hES cells (Line B) without IFN-γ andB2M expression following exposure of WT hES cells with IFN-γ (Line A).The shift (increase) in florescent intensity in the untreated WT hEScells (Line B) as compared to background (shaded region) indicates thatWT hES cells express B2M. The further shift (increase) in florescentintensity beyond WT B2M expression (Line B) following exposure of WT hEScells to IFN-γ suggests that B2M expression increases in WT hES cellsfollowing exposure to IFN-γ (Line A). As such expression of HLA-Class Icell surface proteins can be upregulated upon cellular stress andinflammation such as that caused by at least IFN-γ treatment.

FIG. 2B shows B2M expression in B2M knockout hES cells, knockout cellswere generated using the CRISPR/Cas system. There is substantially noshift (increase) in florescent intensity as compared to the background(shaded region) with or without exposure to IFN-γ suggesting the B2Mknockout hES cells had reduced or eliminated B2M surface expression andthat expression of B2M could not be induced by IFN-γ treatment. In suchB2M knockouts, expression of HLA-Class I cell surface proteins is notupregulated upon cellular stress and inflammation caused by IFN-γtreatment.

This example demonstrates that B2M knockout hES cells had reduced oreliminated B2M surface expression as shown using a B2M antibody.

Example 2: Analysis of Hla Class I Cell Surface Protein Expression in WTand B2M Deficient Cells

Next, wild type and B2M knockout hES cells were analyzed using aPan-HLA-ABC monoclonal antibody (BD Pharmingen, cat#560169) to confirmthat these knockout cells did not express HLA-Class I proteins on thecell surface. The Pan-HLA-ABC antibody reacts with the human majorhistocompatibility complex (MHC) class I proteins, HLA-A, -B, and -C.Expression of Pan-HLA-ABC antibody was assessed in wild type andknockout cells by flow cytometry under normal and inflammatory statesafter exposure to IFN-γ. Normal: without IFN-γ (Line B); inflammatory:exposed to 100 ng/mL of IFN-γ for 18-24 hours (Line A).

FIG. 3A shows Pan-HLA-ABC cell surface protein expression in WT hEScells (Line B) and Pan-HLA-ABC cell surface protein expression followingtreatment of WT hES cells with IFN-γ (Line A). The shift (increase) inflorescent intensity in the untreated WT hES cells (Line B) as comparedto background (shaded region) suggests that WT hES cells expressPan-HLA-ABC. The shift (increase) in florescent intensity beyond WT hESexpression following exposure to IFN-γ suggests that Pan-HLA-ABCexpression increases in WT hES cells following exposure to IFN-γ.

FIG. 3B shows Pan-HLA-ABC cell surface protein expression in B2Mknockout hES cells using the CRISPR/Cas system. There is no shift(increase) in florescent intensity as compared to background (shadedregion) with or without exposure to IFN-γ suggesting the knockouts hadreduced or eliminated HLA-Class I cell surface expression and thatexpression of HLA-Class I proteins was not induced by IFN-γ treatment.

This example demonstrates that B2M knockout hES cells had reduced oreliminated HLA-Class I cell surface expression as shown using aPan-HLA-ABC antibody.

Example 3: Differentiation of B2M Deficient Cells to Pancreatic LineageCells

B2M knockout hES cells were cultured, passaged and proliferated underthe same conditions as WT hES cells as described in Schulz et al. AScalable System for Production of Functional Pancreatic Progenitors fromHuman Embryonic Stem Cells PLoS One 7:5 1-17 (2012) and as described inU.S. Pat. No. 8,895,300 which are both herein incorporated by referencein their entireties. Specifically, Schulz et. al. describe adherent hESCexpansion and suspension-based differentiation.

WT hES cells and B2M−/− hES cells were differentiated in suspensionaggregates using a four (4) stage procedure over the course of about 2weeks (or 14 days) to generate a population of pancreatic cell typesincluding pancreatic progenitors, endocrine progenitors and hormoneexpressing cells, collectively referred to as pancreatic endoderm cells(PEC). Human ES cells were dissociated using accutase and single cellswere aggregated in roller bottles. To initiate differentiation,aggregates were pooled into conical tube(s) and allowed to settle bygravity, followed by a wash using Stage-1 media without growth factors(RPMI+0.2% vol/vol FBS containing 1:5000 dilution ofinsulin-transferrin-selenium (ITS)). The aggregates were re-settled,then resuspended in day 1 media which comprises of RPMI+0.2% vol/vol FBScontaining 1:5000 dilution of insulin-transferrin-selenium (ITS),activin A (100 ng/mL) and wnt3a (50 ng/mL), and distributed to theroller bottles at a density of 2 uL/mL. The roller bottles were placedon FlexiRoll digital cell roller (Argos Technologies) at a speed of 31rpm. Cultures were rotated at about 31 rpm for the remainder of thedifferentiation process with daily media exchange to those described inTable 2 below, adapted from Schulz et al., (2012), supra. Growth,passaging and proliferation of hES is substantially as described in U.S.Pat. Nos. 7,964,402; 8,211,699; 8,334,138; 8,008,07; and 8,153,429. Astandard manufacturing method used for making pancreatic endoderm cells(PEC) derived from human embryonic stem cells is provided below in Table2.

TABLE 2 Standard Manufacturing Method For Making Pancreatic EndodermCells (PEC) Derived From hESC Roller 6-well Time Bottle tray point StageSpeed Speed (day) (1-4) Media Condition (rpm) (rpm) d (−l) hESC XF HA;SP 31 95 Agg. d 0 1 r0.2FBS-ITS1: 5000 A100 W50 31 95 d 1 r0.2FBS-ITS1:5000 A100 31 95 d 2 2 r0.2FBS-ITS1: 1000 K25 IV 31 95 d 3 r0.2FBS-ITS1:1000 K25 31 95 d 4 r0.2FBS-ITS1: 1000 K25 31 105 d 5 3 db-CTT3 N50 31105 d 6 db-CTT3 N50 31 105 d 7 db-CTT3 N50 31 105 d 8 4 db-N50 K50 E5031 105 d 9 db-N50 K50 E50 31 95 d 10 db-N50 K50 E50 31 95 d 11 db-N50K50 E50 31 95 d 12 db-N50 K50 E50 31 95

hESC Agg.: hESC aggregates; XF HA: DMEM/F12 containing GlutaMAX,supplemented with 10% v/v of Xeno-free KnockOut Serum Replacement, 1%v/v non-essential amino acids, 1% v/v penicillin/streptomycin (all fromLife Technologies), 10 ng/mL heregulin-1β (Peprotech) and 10 ng/mLactivin A (R&D Systems); SP: StemPro® hESC SFM (Life Technologies);r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1× GlutaMAX-1 (LifeTechnologies), 1% v/v penicillin/streptomycin; ITS:Insulin-Transferrin-Selenium (Life Technologies) diluted 1:5000 or1:1000; A100: 100 ng/mL recombinant human Activin A (R&D Systems); W50:50 ng/mL recombinant mouse Wnt3A (R&D Systems); K25: 25 ng/mLrecombinant human KGF (R&D Systems); IV: 2.5 μM TGF-β RI Kinaseinhibitor IV (EMD Bioscience); db: DMEM HI Glucose (HyClone)supplemented with 0.5×B-27 Supplement (Life Technologies), 1× GlutaMAX,and 1% v/v penicillin/streptomycin; CTT3: 0.25 μM KAAD-Cyclopamine(Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50: 50ng/mL recombinant human Noggin (R&D Systems); K50: 50 ng/mL recombinanthuman KGF (R&D Systems); E50: 50 ng/mL recombinant human EGF (R&DSystems).

Differentiated B2M −/− and WT PEC were analyzed using flow cytometry todetermine the relative amount of endocrine and pancreatic progenitorcells in the population at stage 4 as shown in Table 3.

TABLE 3 Stage 4 Pancreatic Progenitor Cell Compositions (Percent oftotal Cells) CHGA− NKX6.1− CHGA− PDX1− CHGA− NKX6.1− (Triple CHGA+NKX6.1+PDX1+ PDX1+ negative; (Endo- or − (Pancreatic (PDX+ residual Cellline crine) Progenitors) only) cells) WT 44 42 11 2 B2M−/− clone1 41 525 1 B2M−/− clone2 41 51 7 1 B2M−/− clone3 41 53 5 1

The relative levels of pancreatic endocrine cells, progenitors, PDX-1only cells and triple negative cells in the B2M−/− differentiated cellsin all 3 clones is substantially similar to that observed in WT cells(top row).

This example demonstrates that the B2M−/− hES cells can differentiatedown the pancreatic lineage the same as WT hES cells.

Example 4: Analysis of B2M Expression in WT and B2M Deficient PancreaticEndoderm Cells

Next, wild type and B2M knockout pancreatic endoderm cells (PEC) fromExample 3 were analyzed using flow cytometry without and with IFN-γtreatment: without IFN-γ (Line B); exposed to 100 ng/mL of IFN-γ for18-24 hours (Line A).

FIG. 4A shows B2M expression in WT PEC cells without (Line B) andfollowing treatment with IFN-γ (Line A). The shift (increase) inflorescent intensity in the untreated WT PEC cells (Line B) compared tobackground (shaded region) indicates that WT PEC cells express B2M. Theshift (increase) in florescent intensity beyond WT expression followingexposure of WT PEC to IFN-γ (Line A) indicates that B2M expressionincreases in WT PEC cells following exposure to IFN-γ. That is, exposureto IFN-γ increases B2M expression in WT PEC.

FIG. 4B shows B2M expression in PEC cells derived from B2M knockout hEScells. There is no shift (increase) in florescent intensity as comparedto the background (shaded region) with or without exposure to IFN-γ,suggesting the B2M knockout PEC had reduced or eliminated B2M cellsurface expression and that expression of B2M could not be induced inB2M knockout hES-cell derived PEC by IFN-γ treatment.

This example shows PEC derived from hES cells in which the expression ofB2M was modulated/eliminated had reduced or eliminated B2M surfaceexpression.

Example 5: Analysis of HLA Class I Cell Surface Protein Expression in WTand B2M Knockout Pancreatic Endoderm Cells

Similar to Example 2, wild type and B2M knockout PEC were analyzed usinga Pan-HLA-ABC monoclonal antibody (BD Pharmingen, cat#560169) for HLAClass I cell surface expression. Expression was assessed in wild typeand knockout cells by flow cytometry under two conditions (1) untreated(Line B) and (2) treated with 100 ng/mL of IFN-γ for 18-24 hours (LineA).

FIG. 5A shows HLA-Class I cell surface protein expression in WT PECunder untreated condition (Line B) and expression following treatmentwith IFN-γ (Line A). The shift (increase) in florescent intensity in theuntreated WT PEC (Line B) as compared to the background (shaded region)indicates that WT PEC express HLA-Class I on the cell surface. The shift(increase) in florescent intensity beyond WT PEC expression followingexposure to IFN-γ suggests that HLA Class I cell surface expressionincreases in WT PEC cells following exposure to IFN-γ.

FIG. 5B shows HLA-Class I cell surface protein expression in PEC derivedfrom B2M knockout hES cells. There is no shift (increase) in florescentintensity as compared to the background (shaded region) in PEC cellswith or without exposure to IFN-γ suggesting the knockout had reduced oreliminated HLA surface expression and that expression of HLA Class I nB2M−/− PEC could not be induced by IFN-γ treatment.

As such, reduced or eliminated HLA Class I cell surface expression wasobserved in those PEC cells in which the expression of B2M wasmodulated/eliminated.

Example 6: Analysis of ICAM-1 Cell Surface Protein Expression in WT andB2M Knockout hES Cells

To further define the effect of IFN-γ treatment on target cells, ICAM-1expression in WT and B2M knockout hES cells was assessed by flowcytometry under two conditions: (1) untreated (Line B) and (2) treatedwith 100 ng/mL of IFN-γ for 18-24 hours (Line A). ICAM-1 is required forseveral immunological functions including antigen presentation in targetcells, and is a known NK activating ligand. In vivo, it is possible toderive immunological benefits by disrupting the intercellular ICAM/LFAbinding interaction through the application of specific monoclonalantibodies (“mAbs”), i.e., anti-ICAM-1 or anti-LFA-1. See Isobe et al.,Specific Acceptance of Cardiac Allograft After Treatment With Antibodiesto ICAM-1 and LFA-1, 255 SCIENCE 1125-1127 (February 1992). Applicantsused ICAM-1 antibody from Milteney Biotec Inc., cat#130-103-909.

FIG. 6A shows ICAM-1 protein expression on the cell surface in WT hEScells in untreated (Line B) and following treatment with IFN-γ (Line A).The shift (increase) in florescent intensity as compared to thebackground (shaded region) in untreated WT hES cell (Line B) indicatesthat WT hES cells express ICAM-1 protein on their cell surface. Theshift (increase) in florescent intensity beyond WT expression followingexposure of WT hES cells to IFN-γ suggests that ICAM-1 expressionincreases following exposure to IFN-γ.

FIG. 6B shows ICAM-1 cell surface protein expression in B2M knockout hEScells in untreated (Line B) and following treatment with IFN-γ (Line A).FIG. 6B shows that ICAM-1 cell surface protein expression in the B2Mknockout hES cells was similar to WT hES cells.

This example demonstrates that treatment of WT and B2M knockout hEScells with IFN-γ increases cell surface protein expression of ICAM-1.

Example 7: Analysis of ICAM-1 Cell Surface Protein Expression in WT andB2M Knockout Pancreatic Endoderm Cells

Similar to Examples 4 and 5, wild type and B2M knockout PEC wereanalyzed using an antibody to a known NK activating ligand, e.g. ICAM-1.Cell surface protein expression of ICAM-1 was assessed in WT andknockout PEC by flow cytometry under two conditions (1) untreated and(2) treated with 100 ng/mL of IFN-γ for 18-24 hours.

FIG. 7A shows ICAM-1 cell surface protein expression in WT PEC (Line B)and ICAM-1 cell surface protein expression following treatment of WT PECwith IFN-γ (Line A). The shift (increase) in florescent intensity in theuntreated WT PEC (line B) compared to the background (shaded region)indicates that WT PEC express ICAM-1 protein on their cell surface. Thefurther shift (increase) in florescent intensity beyond WT PECexpression following exposure to IFN-γ suggests that ICAM-1 expressionincreases in WT PEC following exposure to IFN-γ. This demonstrates thatNK activating ligands, and in particular ICAM-1, are highly inducible byIFN-γ stimulation.

FIG. 7B shows ICAM-1 cell surface protein expression in B2M knockoutPEC. ICAM-1 cell surface protein expression in the B2M knockout PEC wassimilar to WT PEC, with and without IFN-γ exposure, with a minorreduction in florescence. Thus, treatment of B2M knockout PEC with IFN-γincreases ICAM-1 cell surface protein expression.

FIG. 8 shows RNA expression array data (Affymetrix), demonstrating thatat the mRNA level, exposure to IFN-γ increases ICAM-1 expression inWThESC, B2M−/− hESC, WT PEC and B2M−/− PEC. Applicants discovered thatcell surface protein expression of NK activating ligand ICAM-1 increasedin the differentiated cell types (WT PEC and B2M−/− PEC) after exposureto IFN-γ Thus, ICAM-1 is highly inducible by IFN-γ stimulation in PEC.

Example 8: Effect of IFN-Gamma Treatment on Additional NK ActivatingLigands

To further characterize the effect of IFN-γ treatment on target cells,WT PEC was assessed by flow cytometry under two conditions (1) untreated(Line B) and (2) treated with 100 ng/mL of IFN-γ for 18-24 hours (LineA) and cell surface protein expression of other known NK activatingligands were analyzed.

FIG. 9 shows CD58 (also known as LFA-3) cell surface protein expressionusing an antibody from BioLegend, cat#330909 in WT PEC untreated (LineB) and following treatment of with IFN-γ (Line A). The large shift(increase) in florescent intensity compared to the background (Line C)in the untreated WT hES cells (Line B) indicates that most of the cellsexpress CD58 protein on the surface. There was a small additional shift(increase) in florescent intensity following exposure to IFN-γ.

FIG. 10 shows CD155 (also known as PVR, NECL-5, HVED) cell surfaceprotein expression using an antibody from Milteneyi Biotech Inc.,cat#130-105-905in WT PEC untreated (Line B) and following treatment withIFN-γ (Line A). The shift (increase) in florescent intensity compared tothe background (Line C) in the untreated WT PEC (Line B) indicates thatWT PEC express CD155. There was not an additional increase (increase) inflorescent intensity beyond untreated condition, following exposure toIFN-γ suggesting that CD155 expression does not increase in WT PECfollowing exposure to IFN-γ.

FIG. 11 shows CEACAM1 (also known as CD66a, BGP, and BGP1) cell surfaceprotein expression using an antibody from Milteneyi Biotech Inc.,cat#130-098-858 in WT PEC untreated (Line B) and following treatment ofwith IFN-γ (Line A). The shift (increase) in florescent intensitycompared to the background (Line C) in the untreated WT PEC (Line B)indicates that WT PEC express CEACAM1 protein on the cell surface. Theshift (increase) in florescent intensity beyond untreated conditionfollowing exposure of WT PEC to IFN-γ suggests that CEACAM1 proteinexpression on cell surface increases in WT PEC following exposure toIFN-γ.

FIG. 12 shows BAT3 (also known as BAG6) cell surface protein expressionusing an antibody from Abcam Inc., cat#ab210838 in WT PEC untreated(Line B) and following treatment with IFN-γ (Line A). The shift(increase) in florescent intensity compared to background (Line C) inthe untreated PEC (Line B) suggests that the cells express CEACAM1.There was not an additional shift (increase) in florescent intensitybeyond untreated condition following exposure of WT PEC to IFN-γsuggests that BAT3 expression does not increase in WT PEC followingexposure to IFN-γ.

FIG. 13 shows CADM1 (also known as NECL2, TSLC1, IGSF4, RA175) cellsurface protein expression using an antibody from MBL internationalCorporation, cat#CM004-4 in WT PEC untreated (Line B) and followingtreatment with IFN-γ (Line A). The shift (increase) in florescentintensity compared to background (Line C) in the untreated condition(Line B) suggests that WT PEC express CADM1 protein on cell surface.There was not an additional shift (increase) in florescent intensitybeyond untreated condition following exposure of PEC to IFN-γ suggeststhat CADM1 expression does not increase following exposure to IFN-γ.

FIG. 14 shows CD112 (also known as Nectin-2, PVRR2, HVEB) expressionusing an antibody from Milteneyi Biotech Inc., cat#130-109-056 in WT PECuntreated (Line B) and following treatment with IFN-γ (Line A). Theshift (increase) in florescent intensity compared to background (Line C)in the untreated condition (Line B), suggests that WT PEC express CD112protein on cell surface. There was not an additional shift (increase) inflorescent intensity beyond untreated condition, following exposure ofPEC to IFN-γ suggesting that CD112 expression does not increasefollowing exposure to IFN-γ.

Example 9: MHC-Class I Deficient, NK Cell-Activating Ligand DeficientCells Prevent NK Cell Mediated Lysis

To test whether the combination of reduced or eliminated HLA-Class Iexpression and reduced or eliminated NK cell-activating ligandexpression is sufficient to prevent NK mediated cell lysis, ICAM-Iexpression was blocked on target cells using ICAM-1 blocking antibody atconcentrations of 5 and 10 ug/mL. Addition of blocking ICAM-1 antibodyto WT or B2M−/− ES cells or PEC caused reduction in NK lysis of targetcells after IFN-γ treatment (FIG. 15).

Staining of Target Cells with Calcein-AM

Calcein release assay is a non-radioactive alternative for studying NKcell cytotoxicity. The target cells take up the fluorescent dye (calceinAM) and cytoplasmically convert it into the active fluorochrome, whichis only released from the cell upon lysis. Lysed cells release thefluorochrome into the supernatant, which is then harvested and theamount of fluorescence quantitated in a fluorometer. The percent celllysis is calculated from the amount of fluorescence present in thesupernatant after incubation in the presence or absence of NK cells(effectors), blocking antibody or both.

Target cells comprised either WT ESC, B2M−/− ESC, WT PEC or B2M−/− PECtreated with or without 100 ng/mL of IFN-γ prior to labelling. Toprepare the target cells, the target cell populations were stained with2 μg/ml Calcein AM staining media (Enzo biosciences 1 mg/mL stocksolution (cat#C3100MP)). The target cells were incubated for 1 hr at 37°C. in 8% CO2 incubator with intermittent mixing. The target cells werewashed twice to remove any free Calcein AM, and resuspended at 1×105cells/ml in RPMI complete media (RPMI, 10% heat inactivated FBS and 1%antibiotics).

Co-Culture Target, NK Cells and Blocking Antibody

Calcein AM labeled target cells were then incubated with NK cells(effector cells) with an effector-to-target ratio (E:T ratio) of 10:1.Specifically, 100 μL of NK cells at a density of 1×106 cells/mL wereadded per well in a 96 well V bottom plate and then 100 μL of Calceinstained ESC or PEC cells were added (1×105 cells/well). Where indicated,blocking antibody to human ICAM-1 surface antigen (R&D Systems, Inc.,cat#BBA3) at concentration of 5 and 10 ug/mL was added to the wells todetermine if NK-mediated cell lysis could be reduced.

Plates were incubated for 4 hours at 37° C. in a 8% CO2 incubator. Afterthe incubation period, plates were centrifuged at 200×g for 2 minutes.100 uL of supernatant was removed carefully and transferred to a blackpigmented 96 well plate and fluorescence measured using a MolecularDevice plate reader (excitation filter: 485 nm/emission filter: 530 nm).Specific lysis was calculated by using the formula, % lysis=100×[(meanfluorescence with antibody−mean spontaneous fluorescence)/(mean maximumfluorescence−mean spontaneous fluorescence)]. Maximum fluorescence wasdetermined by the lysis of cells incubated with detergent (1% TritonX-100) and spontaneous lysis was the fluorescence obtained with targetcells without any antibody or effector cells.

Results

As shown in FIG. 1, the goal is to move from scenario C (NK cells attacktarget cells) to scenario A (no response or reduced response). In FIG.15, this scenario is shown in conditions 4 and 8. In conditions 4 and 8the target cells (B2M−/− hES cells or PEC) lack functional HLA-Class Isurface expression and are exposed to IFN-γ. As discussed above and seenin conditions 4 and 8 exposure to IFN-γ causes an increase in NKactivating ligands (ICAM-1) which results in greater cell lysis comparedto HLA-Class I knockouts without exposure to IFN-γ (compare first barsin condition 3 vs. 4 and condition 7 vs. 8). However, once treated withan ICAM-1 inhibitory antibody which serves to block expression of the NKactivating signal on the target cell, NK mediated cell lysis falls from83% to 73% in B2M−/− hES cells and 72% to 61% in B2M−/− PEC treated withIFN-γ. The percentage NK mediated cell lysis does not drop to zerobecause, ICAM-1 cell surface protein expression may not be completelyblocked using the blocking antibody and as discussed above, the targetcells express more than one NK activating ligand. The percentage of NKmediated cell lysis is expected to fall to a greater extent when ICAM-1expression is inhibited further and other NK activating ligands areblocked in the target cells. Therefore, the ICAM-1 inhibitory antibodycan be combined with additional NK activating ligand inhibitoryantibodies including inhibitory antibodies to any of the ligands listedin Table 1 and preferably those in category 1 (Known activating ligands)and 2 (Potential candidates for activating ligands identified from genechip data, those are upregulated in PEC and/or ESC after IFNγ). In oneembodiment, the ICAM1 gene and other NK activating ligand genes aredisrupted in order to completely block their activity.

Scenario D from FIG. 1 is presented in the first bars of conditions 2and 6 of FIG. 15. In conditions 2 and 6, the WT hES cells and PEC haveupregulated cell surface protein expression of both HLA-Class I antigensand NK activating ligands as a result of their exposure to IFN-γ. Whenthe cells are incubated with the ICAM-1 inhibitory antibody, thisrepresents a situation of moving from Scenario D towards Scenario B ofFIG. 1. Upon incubation with ICAM1 inhibitory antibody, cell deathdecreases: 79% to 60% in WT hES cells and 53% to 27% in WT PEC exposedto IFN-γ. Indeed, when WT PEC is exposed to IFN-γ and incubated with theICAM-1 inhibitory antibody, NK cell lysis falls below that of untreatedWT PEC: 27% for WT PEC, IFN-γ and ICAM-1 inhibitory antibody compared to37% WT PEC, ICAM-1 inhibitory antibody with no IFN-γ.

Scenario B from FIG. 1 is exemplified by the ICAM1 antibody treated barsin conditions 1, 2, 5 and 6 in FIG. 15. There, the target hES cells andPEC have HLA-Class I and NK activating ligands, but when the targetcells are not exposed to IFN-γ, there is no increase in cell surfaceprotein expression of ICAM-1. As a result, the ICAM-1 inhibitoryantibody has less of an effect. Hence, cell lysis remains about thesame: 58% to 57% in hES cells and 33% to 37% in PEC cells.

Scenario C from FIG. 1 is similar to the first bars in conditions 3, 4,7 and 8 in FIG. 15. In these conditions, the cells have no HLA-Class Icell surface expression as a result of B2M−/−. Therefore, when NKactivating ligands are not activated by IFN-γ exposure of the cells tothe ICAM-1 inhibitory antibody has little effect. 71% to 70% in hEScells and 54% to 57% in PEC.

As a general observation, NK mediated cell lysis is less indifferentiated cell populations (WT PEC and B2M−/− PEC) compared toundifferentiated cell populations (WT hES and B2M−/− hES). NK mediatedcell lysis increases to a greater extent when cells (WT or B2M −/−knockout, hES or PEC) are activated with IFN-γ.

Example 10: Generation of NK Activating Ligand Deficient B2M−/− KnockouthES Cells

Example 9 describes that inhibiting or quenching ICAM-1 expressionprotects the IFN-γ treated B2M −/− PEC from NK mediated cell killingactivity. Hence, to protect B2M−/− PEC from NK cell mediated killingpost-transplant, it will be desirable to disrupt NK cell activatingligand genes. For example, based on the examples above, ICAM-1, a knownNK activating ligand gene can be disrupted or ‘knocked out’. Preferably,both alleles of the ICAM-1 locus in the B2M−/− CyT49 hESC line can bedisrupted using CRISPR/Cas9 or any other gene editing technology nowknown or in the future should be known See PCT Publication No.WO2016183041A, which is incorporated herein by reference in itsentirety. Examples of published sequences for ICAM-1 are submitted asSEQ ID NOS.: 4, 5, and 6. For example, in one embodiment of theinvention, NEXTGEN™ CRISPR (Transposagen Inc., Lexington Ky.), whichincorporates dual guide RNA's and a catalytically inactive Cas9 proteinfused to the FokI nuclease, is used to gene edit the cells.

Plasmids containing the guide RNAs and Cas9 can be electroporated intoB2M−/− CyT49 hESC, and seeded onto tissue culture plates.Post-electroporation, cells can be sorted for negative reactivity to anICAM-1 antibody by flow cytometry. Sorted cells can be plated at clonaldensity. Individual clones can be picked and re-plated. Clones can beexpanded and cryopreserved. Expanded clones that showed a normalkaryotype by G-banding, and are negative for expression of ICAM-1protein and for surface expression of ICAM-1 proteins by flow cytometryand/or immunofluorescence can be chosen for further experimentation.

As described above, cells deficient for ICAM1 and B2M cannot express atleast one NK activating ligand and at least one or all MHC-Class Iprotein on their cell surface and therefore should not bind to NK cellactivating receptors and are protected from NK mediated cell death.

Example 11: Generation of Multiple NK Activating Ligand Deficient B2M−/−Knockout hES Cells

Example 9 and 10 demonstrate that inhibiting functional cell surfaceexpression (anti-NK activating ligand) and gene disruption of NK cellactivating ligand (e.g., ICAM1−/−) in combination with a disruption ofMHC-Class I cell surface expression (e.g., B2M−/−) can provide targetcells protection from NK mediated cell death. Transplantation of a celldeficient in more than one NK cell activating ligand can be produced andconfer further protection from NK mediated cell death.

The CD58 gene is selected as the NK activating ligand gene to knockout.Both alleles of the CD58 locus can be disrupted using CRISPR/Cas9technology, using known techniques as outlined in WO2016183041A, in theB2M−/−:ICAM−/− double knockout CyT49 hESC line. Examples of publishedsequences for CEACAM1 are submitted as SEQ ID NOS.: 7, 8, and 9. Again,the version of editing can be NEXTGEN™ CRISPR (Transposagen Inc.,Lexington Ky.).

Plasmids containing the guide RNAs and Cas9 can be electroporated intoB2M−/−, ICAM−/− knockout CyT49 hESC, and seeded onto tissue cultureplates. Post-electroporation, cells can be sorted for negativereactivity to a CD58 antibody by flow cytometry. Sorted cells can beplated at clonal density. Individual clones can be picked and re-plated.Clones can be expanded and cryopreserved. Expanded clones that showed anormal karyotype by G-banding, and are negative for expression of CD58protein and for surface expression of CD58 proteins by flow cytometryand/or immunofluorescence can be chosen for further experimentation.

As described above, cells with disrupted, deleted or modified ICAM1,CD58 and B2M cannot express ICAM1, CD58 nor MHC Class I proteins ontheir cell surface and therefore should not bind to NK cell activatingreceptors and are protected from NK mediated cell death.

What is claimed is:
 1. A method of inhibiting cellular graft rejectionof pancreatic endoderm cells (PEC) in an immunocompetent orimmunosuppressed human, comprising: transplanting a cell encapsulationdevice comprising a cell population comprising PEC that are CHGA⁻,NKX6.1⁺, PDX1⁺ pancreatic progenitor cells, and PEC that are CHGA⁺pancreatic endocrine cells, into a location in the immunocompetent orimmunosuppressed human that permits host vascularization, wherein thefunction of at least one major histocompatibility complex (MHC)-Class Igene and at least one Natural killer (NK) cell activating ligand isdisrupted or inhibited in the cell population, resulting in reducedbinding of the NK activating ligand to a corresponding NK activatingreceptor, and the disruption or inhibition of the at least one MHC-ClassI gene and the at least one NK cell activating ligand results ininhibiting cellular graft rejection of the cell population in theimmunocompetent or immunosuppressed human.
 2. The method of claim 1,wherein the location is subcutaneous in the immunocompetent orimmunosuppressed human.
 3. The method of claim 1, wherein the MHC-ClassI gene codes for beta-2 microglobulin (B2M) or codes for a humanleucocyte antigen (HLA)-ABC cell surface protein.
 4. The method of claim1, wherein the at least one NK cell activating ligand is: a)Intercellular Adhesion Molecule 1 (ICAM1), CD58, CD155, CarcinoembryonicAntigen-Related Cell Adhesion Molecule 1 (CEACAM1), Cell AdhesionMolecule 1 (CADM1), MHC-Class I Polypeptide-Related Sequence A (MICA),or MHC-Class I Polypeptide-Related Sequence B (MICB); b) ICAM1 and CD58;c) ICAM-1, CD58, and CD155; d) ICAM1, CD58, CD155, and CADMI; e) CD58and CADM1; or f) ICAM-1, CADM1, and CD155.
 5. The method of claim 1,wherein the cell population further expresses a protein which whenexpressed in the presence of a cell death inducing agent, the cell deathinducing agent is capable of killing cells in the cell population. 6.The method of claim 1, wherein the human is an immunocompetent human. 7.The method of claim 1, wherein the human is an immunosuppressed human.8. A method of producing insulin in an immunocompetent orimmunosuppressed human, comprising: transplanting a cell encapsulationdevice comprising a cell population comprising: pancreatic endodermcells (PEC) that are CHGA⁻, NKX6.1⁺, PDX1⁺ pancreatic progenitor cells,and PEC that are CHGA⁺ pancreatic endocrine cells, into a location inthe immunocompetent or immunosuppressed human that permits hostvascularization, wherein the function of at least one majorhistocompatibility complex (MHC)-Class I gene and at least one Naturalkiller (NK) cell activating ligand is disrupted or inhibited in the cellpopulation, resulting in reduced binding of the NK activating ligand toa NK activating receptor, and wherein the cell population matures in theimmunocompetent or immunosuppressed human into a mature cell populationthat produces insulin in response to glucose stimulation.
 9. The methodof claim 8, wherein the location is subcutaneous in the immunocompetentor immunosuppressed human.
 10. The method of claim 8, wherein theMHC-Class I gene codes for beta-2 microglobulin (B2M) or codes for ahuman leucocyte antigen (HLA)-ABC cell surface protein.
 11. The methodof claim 8, wherein the at least one NK cell activating ligand is: a)Intercellular Adhesion Molecule 1 (ICAM1), CD58, CD155, CarcinoembryonicAntigen-Related Cell Adhesion Molecule 1 (CEACAM1), Cell AdhesionMolecule 1 (CADM1), MHC-Class I Polypeptide-Related Sequence A (MICA),or MHC-Class I Polypeptide-Related Sequence B (MICB); or b) ICAM1 andCD58; c) ICAM-1, CD58, and CD155; d) ICAM1, CD58, CD155, and CADMI; e)CD58 and CADM1; or f) ICAM-1, CADM1, and CD155.
 12. The method of claim8, wherein the cell population further expresses a protein which whenexpressed in the presence of a cell death inducing agent, the cell deathinducing agent is capable of killing cells in the cell population. 13.The method of claim 8, wherein the human is an immunocompetent human.14. The method of claim 8, wherein the human is an immunosuppressedhuman.